RESUMEN
The mammalian target of rapamycin (mTOR) is a central regulator of G1 cell cycle protein synthesis that precedes commitment to normal cellular replication. We have studied the effect of cell cycle inhibitor-779 (CCI-779), a rapamycin ester that inhibits mTOR function, on the proliferation of a panel of breast cancer cell lines. Six of eight lines studied were sensitive (IC(50)< or = 50 nM) and two lines were resistant (IC(50)>1.0 microM) to CCI-779. Sensitive lines were estrogen dependent (MCF-7, BT-474, T-47D), or lacked expression of the tumor suppressor PTEN (MDA-MB-468, BT-549), and/or overexpressed the Her-2/neu oncogene (SKBR-3, BT-474). Resistant lines (MDA-MB-435, MDA-MB-231) shared none of these properties. CCI-779 (50 nM) inhibited mTOR function in both a sensitive and a resistant line. In nu/nu mouse xenografts, CCI-779 inhibited growth of MDA-MB-468 (sensitive) but not MDA-MB-435 resistant tumors. Treatment of sensitive lines with CCI-779 resulted in a decrease in D-type cyclin and c-myc levels and an increase in p27(kip-1) levels. There was good correlation between activation of the Akt pathway and sensitivity to CCI-779. Amplification of mTOR-regulated p70S6 kinase, which is downstream of Akt, may also have conferred CCI-779 sensitivity to MCF-7 cells. Taken together, the data suggest that mTOR may be a good target for breast cancer therapy, especially in tumors with Akt activation resulting from either growth factor dependency or loss of PTEN function.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Inhibidores de Proteínas Quinasas , Proteínas Quinasas , Sirolimus/análogos & derivados , Sirolimus/farmacología , Animales , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Evaluación Preclínica de Medicamentos , Resistencia a Antineoplásicos , Femenino , Ratones , Ratones Desnudos , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
A series of 4-anilino-3-cyano-6,7-dialkoxyquinolines with different substituents attached to the 4-anilino group has been prepared that are potent MEK (MAP kinase kinase) inhibitors. The best activity is obtained when a phenyl or a thienyl group is attached to the para-position of the aniline through a hydrophobic linker, such as an oxygen, a sulfur, or a methylene group. The most active compounds show low nanomolar IC(50)'s against MEK (MAP kinase kinase), and have potent growth inhibitory activity in LoVo cells (human colon tumor line).
Asunto(s)
Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinolinas/farmacología , Compuestos de Anilina/química , Antineoplásicos/química , División Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Humanos , Quinolinas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
A series of 3-cyano-4-(phenoxyanilino)cyanoquinolines has been prepared as MEK (MAP kinase kinase) inhibitors. The best activity is seen with alkoxy groups at both the 6- and 7-positions. The lead compounds show low nanomolar IC50's against MAP kinase kinase, and have potent inhibitory activity in tumor cells.
Asunto(s)
Antineoplásicos/síntesis química , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Nitrilos/farmacología , Quinolinas/farmacología , Compuestos de Anilina/síntesis química , Compuestos de Anilina/farmacología , Antineoplásicos/farmacología , División Celular/efectos de los fármacos , Neoplasias del Colon/patología , Ensayo de Inmunoadsorción Enzimática , Humanos , Concentración 50 Inhibidora , Nitrilos/síntesis química , Quinolinas/síntesis química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Cytosolic free Ca2+ concentration ([Ca2+]i) was measured in differentiated PC12 cells to test whether chemical hypoxia selectively alters intracellular Ca2+ in growth cones and cell bodies. Hypoxia increased [Ca2+]i and exaggerated its response to K+ depolarization in both parts of the cells. [Ca2+]i in the cell bodies was greater than that in the growth cones under resting conditions and in response to K+ or hypoxia. Ca2+-channel blockers selectively altered these responses. The L-channel blocker nifedipine reduced [Ca2+]i following K+ depolarization by 67% in the cell bodies but only 25% in the growth cones. In contrast, the N-channel blocker omega-conotoxin GVIA (omega-CgTX) diminished K+-induced changes in [Ca2+]i only in the growth cones. During hypoxia, nifedipine was more effective in the cell bodies than in the growth cones. During hypoxia, omega-CgTX diminished K+-induced changes by 50-75% in both parts of the cell, but only immediately after depolarization. The combination of nifedipine and omega-CgTX diminished the [Ca2+]i response to K+ with or without hypoxia by >90% in the cell body and 70% in the growth cones. Thus, the increased Ca2+ entry with K+ during hypoxia is primarily through L channels in the cell bodies, whereas in growth cones influx through L and N channels is about equal. The results show that chemical hypoxia selectively alters Ca2+ regulation in the growth cone and cell body of the same cell.
Asunto(s)
Calcio/metabolismo , Diferenciación Celular , Hipoxia de la Célula , Factores de Crecimiento Nervioso/farmacología , Células PC12/ultraestructura , Animales , Bloqueadores de los Canales de Calcio/farmacología , Interacciones Farmacológicas , Nifedipino/administración & dosificación , Nifedipino/farmacología , Células PC12/metabolismo , Péptidos/administración & dosificación , Péptidos/farmacología , Potasio/farmacología , Ratas , omega-Conotoxina GVIARESUMEN
The experiments in this paper identify multiple calcium compartments in cultured human fibroblasts and reveal abnormalities in one of these pools in cells from Alzheimer patients. In the presence of external calcium, bradykinin (BK) increased cytosolic free calcium ([Ca2+]i) about 3-fold and then [Ca2+]i rapidly declined. Omission of calcium from the media did not affect the BK-induced peak, which indicates that the peak reflects internal stores. Other compounds that also released calcium from internal stores included A23187 (a calcium ionophore), thapsigargin (Tg; an inhibitor of endoplasmic reticulum ATPase), and FCCP (an uncoupler of oxidative phosphorylation). The [Ca2+]i response to sequential addition of compounds in calcium-free media identified discrete internal calcium stores. BK depleted internal calcium pools such that subsequent stimulation with BK, FCCP or bombesin did not increase [Ca2+]i. However, A23187 or thapsigargin still elicited responses. A23187 depleted essentially all internal calcium pools. Either Tg or FCCP reduced the calcium stores that could be released by BK or A23187. Thus, cellular calcium compartments that respond to BK and A23187 partially overlap. The common pool includes Tg-and FCCP-sensitive compartments. Calcium stores were examined in cells from Alzheimer disease patients, because previous studies suggest that their calcium homeostasis is altered. A23187 addition to BK-treated cells produced a 95% greater response in cell lines from Alzheimer patients (n = 7) than in those from controls (n = 5). Thus, various calcium stores can be pharmacologically distinguished in fibroblasts and at least one of these compartments is abnormal in Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Calcio/metabolismo , Bradiquinina/farmacología , Calcimicina/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Compartimento Celular , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Humanos , Ionóforos/farmacología , Terpenos/farmacología , TapsigarginaRESUMEN
Calcium homeostasis and mitochondrial oxidative metabolism interact closely in brain and both processes are impaired during hypoxia. Since the regulation of the pyruvate dehydrogenase complex (PDHC) may link these two processes, the relation of cytosolic free calcium ([Ca2+]i) to the activation state of PDHC (PDHa) was assessed in isolated nerve terminals (i.e. synaptosomes) under conditions that alter [Ca2+]i. K+ depolarization elevated [Ca2+]i and PDHa and both responses required external calcium. Treatment with KCN, an in vitro model of hypoxia decreased ATP and elevated [Ca2+]i and PDHa. Furthermore, in the presence of KCN, PDHa became more sensitive to K+ depolarization as indicated by larger changes in PDHa than in [Ca2+]i. The calcium ionophore Br-A23187 elevated [Ca2+]i, but did not affect PDHa. K+ depolarization elevated [Ca2+]i and PDHa even if [Ca2+]i was elevated by prior addition of ionophore or KCN. Previous in vivo studies by others show that PDHa is altered during and after ischemia. The current in vitro results suggest that hypoxia, only one component of ischemia, is sufficient to increase PDHa. These data also further support the notion that PDHa is regulated by [Ca2+]i as well as by other factors such as ATP. Our results are consistent with the concept that PDHa in nerve endings may be affected by [Ca2+]i and that these two processes are clearly linked.
Asunto(s)
Calcio/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Sinaptosomas/enzimología , Animales , Calcimicina/farmacología , Citosol/metabolismo , Ácido Egtácico/farmacología , Activación Enzimática , Homeostasis , Cinética , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos , Cloruro de Potasio/farmacología , Cianuro de Potasio/farmacología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/fisiologíaRESUMEN
Understanding the cellular response to hypoxia may help elucidate the role of altered oxidation in neuronal death or abnormal cell function. In PC12 cells, 30 min of chemical hypoxia (i.e., KCN) reduced ATP concentrations by 92%, but diminished viability by only 10%. Ten minutes of hypoxia increased cytosolic free calcium ([Ca2+]i) 2.5-fold above control, but after 30 min of hypoxia, [Ca2+]i was slightly below that of nonhypoxic cells. Short periods of hypoxia also exaggerated the K(+)-induced elevation of [Ca2+]i, but by 30 min these ATP-depleted cells reestablished a calcium gradient that was equal to nonhypoxic, K(+)-depolarized cells. Thus, 30 min of severe ATP depletion left [Ca2+]i and viability relatively unaffected. Nerve growth factor caused slight, but significant, improvements in ATP and viability of hypoxic cells, but had no effect on [Ca2+]i. Although [Ca2+]i was equivalent in control and hypoxic cells after 30 or 60 min, hypoxia abolished the K(+)-stimulated elevation of [Ca2+]i. The nerve growth factor induction of c-fos, an indicator of the genomic response, was diminished by approximately 80%. Thus, hypoxic PC12 cells with greatly reduced ATP stores maintained normal [Ca2+]i, but their ability to respond to external stimulation was impaired. Further, the reduced oxidation that occurs in the brain in a variety of pathological conditions may interfere with the cellular response to stimulation and growth factors.
Asunto(s)
Calcio/metabolismo , Citosol/metabolismo , Regulación Neoplásica de la Expresión Génica , Hipoxia/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Citosol/química , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hipoxia/genética , Factores de Crecimiento Nervioso/farmacología , Células PC12 , RatasRESUMEN
The precise mechanism by which altered oxidative metabolism impairs neuronal function is unknown. Previous indirect studies suggest that anoxia's effects on the mitochondrial membrane potentials may underlie anoxia's actions. Twenty minutes of anoxia reduced the mitochondrial membrane potential of intact synaptosomes by 38-59 mV, but diminished the plasma membrane potential by only 4-10 mV. Anoxia did not alter the response of the plasma or mitochondrial membrane potentials to K+, nor did anoxia affect the reaction of the plasma membrane potential to valinomycin. However, anoxia diminished the response of the mitochondrial membrane potential to valinomycin by 50%. Thus, partial collapse of the mitochondrial membrane potential may be an important mediator of hypoxia-or anoxia-induced changes in neuronal function.
Asunto(s)
Encéfalo/fisiología , Membranas Intracelulares/fisiología , Mitocondrias/fisiología , Sinaptosomas/fisiología , Anaerobiosis , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Hipoxia , Membranas Intracelulares/efectos de los fármacos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Potasio/farmacología , Ratas , Sinaptosomas/efectos de los fármacos , Valinomicina/farmacologíaRESUMEN
Altered cellular calcium homeostasis may be important in the pathophysiology of aging and Alzheimer's disease. Calcium transport by freshly isolated lymphocytes declines with Alzheimer's disease compared with age-matched controls. To determine if these changes occur in the absence of complications due to drugs, diet or any of the other variables that are dependent upon the state of the patients, cytosolic free calcium ([Ca2+]i) was determined in cultured lymphoblasts from young and aged control subjects, as well as from Alzheimer patients. Lymphoblast [Ca2+]i was determined with the fluorescent probe fura-2 in either the presence or absence of serum. In cells that were grown in serum free medium, neither growth rates nor [Ca2+]i varied between groups. Growing cells in serum containing medium doubled growth rates and [Ca2+]i. However, [Ca2+]i from young, aged and Alzheimer groups were still similar. Thus, an age- or Alzheimer-related alteration in [Ca2+]i does not occur in cultured lymphoblasts.
Asunto(s)
Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Calcio/metabolismo , Adulto , Anciano , Transporte Biológico Activo , Línea Celular , Citosol/metabolismo , Femenino , Fura-2 , Homeostasis , Humanos , Activación de Linfocitos , Linfocitos/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Several studies suggest that alterations in the receptor-mediated phosphoinositide cascade and cytosolic free calcium concentration ([Ca2+]i) are involved in the pathophysiology of aging and Alzheimer's disease. Therefore, the phosphoinositide cascade and [Ca2+]i were determined under resting conditions and after stimulation with bradykinin (100 nM) in cultured human skin fibroblasts from young (21 +/- 3 years), normal aged (59 +/- 6 years) and Alzheimer subjects (58 +/- 6 years). The inositol polyphosphates (IP3, IP2 and IP) were monitored after prelabeling the cells with [3H]inositol in serum free medium. [Ca2+]i was determined with the fluorescent probe, fura-2AM, under exactly analogous conditions. The bradykinin-induced formation of IP3 and IP2 increased significantly in fibroblasts from normal aged and Alzheimer donors compared to young subjects, but did not differ from each other. Bradykinin-induced IP3 formation was 63-117% above the young group at time points between 10-60 s in normal aged or Alzheimer donors. Bradykinin-induced IP2 formation was 49-59% above the young group at time points between 10-60 s in normal aged or Alzheimer subjects. Neither the basal [Ca2+]i, nor the bradykinin-stimulated [Ca2+]i, differed among fibroblasts from young, normal aged and Alzheimer donors. The precise molecular basis and pathophysiological significance of the enhanced bradykinin-induced phosphoinositide cascade in fibroblasts from aged donors remains to be determined.
Asunto(s)
Envejecimiento/metabolismo , Enfermedad de Alzheimer/metabolismo , Bradiquinina/farmacología , Calcio/metabolismo , Fosfatos de Inositol/metabolismo , Adulto , Anciano , Fibroblastos/efectos de los fármacos , Humanos , Persona de Mediana Edad , Estimulación QuímicaRESUMEN
Altered cytosolic free calcium concentrations ([Ca2+]i) accompany impaired brain metabolism and may mediate subsequent effects on brain function and cell death. The current experiments examined whether hypoxia-induced elevations in [Ca2+]i are from external or internal sources. In the absence of external calcium, neither KCl depolarization, histotoxic hypoxia (KCN), nor the combination changed [Ca2+]i. However, with external CaCl2 concentrations as small as 13 microM, KCl depolarization increased [Ca2+]i instantaneously while hypoxia gradually raised [Ca2+]i. The combination of KCN and KCl was additive. Increasing external calcium concentrations up to 2.6 mM exaggerated the effects of K+ and KCN on [Ca2+]i, but raising medium calcium to 5.2 mM did not further augment the rise. Diminishing the sodium in the media, which alters the activity and perhaps the direction of the Na/Ca exchanger, reduced the increase in [Ca2+]i due to hypoxia, but enhanced the KCl response. The changes in ATP following K+ depolarization, KCN or their combination in the presence of physiological calcium concentrations did not parallel alterations in [Ca2+]i, which suggests that diminished activity of the calcium dependent ATPase does not underlie the elevation in [Ca2+]i. Valinomycin, an ionophore which reduces the mitochondrial membrane potential, elevated [Ca2+]i and the effects were additive with K+ depolarization in a calcium dependent manner that paralleled the effects of hypoxia. Together these results suggest that hypoxia-induced elevations of synaptosomal [Ca2+]i are due to an inability of the synaptosome to buffer entering calcium.
Asunto(s)
Calcio/metabolismo , Corteza Cerebral/metabolismo , Sinaptosomas/metabolismo , Animales , Calcio/farmacología , Hipoxia de la Célula , Citosol/metabolismo , Cinética , Masculino , Ratones , Ratones Endogámicos , Cloruro de Potasio/farmacología , Cianuro de Potasio/farmacología , Sinaptosomas/efectos de los fármacos , Sinaptosomas/fisiología , Factores de Tiempo , Valinomicina/farmacologíaRESUMEN
The inositol triphosphate (IP3) that results from hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) is generally accepted to be responsible for the mobilization of intracellular calcium. However, some studies suggest that low concentrations of agonists elevate cytosolic free calcium concentration ([Ca2+]i) without IP3 formation. Thus, in the present studies, a comparison of the temporal response of inositol phosphates (IP3, IP2 and IP) and [Ca2+]i to a wide range of bradykinin concentrations was used to examine the relation of these two signal transduction events in cultured human skin fibroblasts (GM3652). In addition, the effects of alterations in internal or external calcium on the response of these second messengers to bradykinin were determined. Bradykinin stimulated accumulation of inositol phosphates and a rise of [Ca2+]i in a time- and dose-dependent manner. Decreasing the bradykinin concentration from 1 microM to 0.1 microM increased the time until the IP3 peak, and when the bradykinin concentration was reduced to 0.01 microM IP3 was not detected. [Ca2+]i was examined under parallel conditions. As the bradykinin concentration was reduced from 1 microM to 0.01 microM, the time to reach the peak of [Ca2+]i increased progressively, but the magnitude of the peak was unaltered. These two second messengers were variably dependent on external calcium. Although the bradykinin-stimulated initial spike of [Ca2+]i did not depend on extracellular calcium, the subsequent sustained levels of [Ca2+]i were abolished in calcium free medium. The bradykinin-stimulated inositol phosphate formation was not dependent on the extracellular calcium nor on the elevation of [Ca2+]i that was produced with Br-A23187. These results demonstrate that bradykinin-induced IP3 formation can be independent of [Ca2+]i and of external calcium, whereas changes in [Ca2+]i are partially dependent on external calcium.
Asunto(s)
Bradiquinina/farmacología , Calcio/metabolismo , Fosfatos de Inositol/metabolismo , Piel/metabolismo , Calcio/farmacología , Línea Celular , Citosol/efectos de los fármacos , Citosol/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Fosfatos de Inositol/aislamiento & purificación , Cinética , Factores de TiempoRESUMEN
Elevations in cytosolic free calcium ([Ca2+]i) precede electrophysiological alterations due to ischemia in vivo. An in vitro model of these changes would help to elucidate their molecular basis. A model of postdecapitative ischemia was used to study these interactions. Nerve endings (i.e. synaptosomes) were isolated either immediately after decapitation or at various time periods after decapitation. Synaptosomal [Ca2+]i and ATP concentrations were determined during a basal period and following depolarization. K(+)-depolarization produced an initial spike of [Ca2+]i that was followed by a new equilibrium value. Ischemia elevated the basal [Ca2+]i and the new equilibrium [Ca2+]i after KCl but suppressed the [Ca2+]i spike. However, the difference between the basal [Ca2+]i and the new equilibrium [Ca2+]i after K(+)-depolarization did not vary with ischemia. Although ischemia reduced ATP, K(+)-depolarization did not alter ATP concentrations in either the controls or the ischemia group, which suggests that synaptosomal mitochondria can meet an energy demand after ischemia. ATP was inversely related to the basal or the new equilibrium [Ca2+]i following depolarization. These changes in [Ca2+]i may underlie the alterations in neurotransmitter release and cell death following ischemia. This appears to be a useful model in which to study the molecular basis of ischemia induced changes in [Ca2+]i.
Asunto(s)
Isquemia Encefálica/metabolismo , Calcio/metabolismo , Sinaptosomas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Citosol/metabolismo , Homeostasis , Masculino , Potenciales de la Membrana , Ratones , Ratones Endogámicos , Potasio/farmacologíaRESUMEN
Exposing brain slices to reduced oxygen tensions or impairing their ability to utilize oxygen with KCN decreases acetylcholine (ACh) but increases dopamine (DA) and glutamate in the medium at the end of a release incubation. To determine if these changes are due to alterations in the presynaptic terminals, release from isolated nerve endings (i.e. synaptosomes) was determined during histotoxic hypoxia (KCN). KCN reduced potassium-stimulated synaptosomal ACh release and increased dopamine and glutamate release. Since several lines of evidence suggest that altered calcium homeostasis underlies these changes in release, the effects of reducing medium calcium concentrations from 2.3 to 0.1-mM were determined. In low calcium medium, KCN still increased dopamine and glutamate release, but had no effect on ACh release. Hypoxia increased cytosolic-free calcium in both the normal and low calcium medium, although the elevation was less in the low calcium medium. Thus, the effects of histotoxic hypoxia on cytosolic free calcium concentration paralleled those on glutamate and dopamine release. Reducing the glucose concentration of the medium also increased cytosolic-free calcium. The data are consistent with the hypothesis that hypoxia and hypoglycemia increase cytosolic-free calcium, which stimulates the release of dopamine and glutamate, whose excessive release may lead to subsequent cellular damage postsynaptically.