RESUMEN
Exposure to the chemical warfare nerve agent VX is extremely toxic, causing severe cholinergic symptoms. If not appropriately treated, death ultimately ensues. Based on our previously described whole-body vapor exposure system, we characterized in detail the clinical outcome, including respiratory dynamics, typical of whole-body exposure to lethal doses of VX vapor in freely moving rats. We further evaluated the efficacy of two different antidotal regimens, one comprising a single and the other repeated administration of antidotes, in countering the toxic effects of the exposure. We show that a 15 min exposure to air VX concentrations of 2.34-2.42 mg/m3 induced a late (15-30 min) onset of obvious cholinergic signs, which exacerbated over time, albeit without convulsions. Marked eye pathology was observed, characterized by pupil constriction to pinpoint, excessive lacrimation with red tears (chromodacryorrhea) and corneal damage. Respiratory distress was also evident, characterized by a three-fourfold increase in Penh values, an estimate of lung resistance, and by lung and diaphragm histological damage. A single administration of TAB (the oxime TMB-4, atropine and the anticholinergic and antiglutamatergic benactyzine) at the onset of clinical signs afforded only limited protection (66% survival), with clinical deterioration including weight loss, chromodacryorrhea, corneal damage, increased airway resistance and late death. In contrast, a combined therapy of TAB at the onset of clinical signs and repeated administration of atropine and toxogonin (ATOX) every 3-5 h, a maximum of five i.m. injections, led to 100% survival and a prompt recovery, accompanied by neither the above-described signs of eye pathology, nor by bronchoconstriction and respiratory distress. The necessity of recurrent treatments for successful elimination of VX vapor toxicity strongly supports continuous penetration of VX following termination of VX vapor exposure, most likely from a VX reservoir formed in the skin due to the exposure. This, combined with the above-described eye and respiratory pathology and absence of convulsions, are unique features of whole-body VX vapor exposure as compared to whole-body vapor exposure to other nerve agents, and should accordingly be considered when devising optimal countermeasures and medical protocols for treatment of VX vapor exposure.
Asunto(s)
Antídotos/administración & dosificación , Atropina/administración & dosificación , Benactizina/administración & dosificación , Sustancias para la Guerra Química/toxicidad , Compuestos Organotiofosforados/toxicidad , Trimedoxima/administración & dosificación , Animales , Antídotos/farmacología , Atropina/farmacología , Benactizina/farmacología , Inhibidores de la Colinesterasa/administración & dosificación , Inhibidores de la Colinesterasa/toxicidad , Esquema de Medicación , Combinación de Medicamentos , Exposición a Riesgos Ambientales/efectos adversos , Oftalmopatías/inducido químicamente , Oftalmopatías/prevención & control , Masculino , Cloruro de Obidoxima/administración & dosificación , Compuestos Organotiofosforados/administración & dosificación , Ratas , Ratas Sprague-Dawley , Enfermedades Respiratorias/inducido químicamente , Enfermedades Respiratorias/prevención & control , Trimedoxima/farmacologíaRESUMEN
VX, a potent inhibitor of cholinesterase (ChE), is considered as one of the most toxic, persistent and least volatile nerve agents. VX is absorbed in various environmental surfaces and is gradually released long after its initial dispersal. Its toxicity is mainly caused by disrupting central and peripheral cholinergic nervous system activity, leading to potential long-term detrimental effects on health. The primary objective of the present study was to assess the threshold VX dose leading to minimal physiological alterations following prolonged VX exposure. Characterization of such a threshold is crucial for dealing with unresolved operative dilemmas such as when it is safe enough to resettle a population that has been evacuated from a VX-contaminated area. Rats, continuously exposed to various doses of VX (0.225-45 µg/kg/day) for 4 weeks via implanted mini-osmotic pumps, showed a dose-dependent and continuous decrease in ChE activity in whole blood, brain and muscles, ranging between 20 and 100%. Exposure to 13.5 µg/kg/day led to a stable low ChE activity level (~ 20%), accompanied by transient and negligible electrocorticogram spectral power transformations, especially in the theta and alpha brain wave frequencies, and a significant decrease in total brain M2 receptor density. These changes were neither accompanied by observable signs of intoxication nor by changes in motor function, circadian rhythm or TSPO level (a reliable marker of brain damage). Following exposure to lower doses of 2.25 and 0.225 µg/kg/day, the only change measured was a reduction in ChE activity of 60 and 20%, respectively. Based on these results, we delineate ChE inhibition as the physiological measure most susceptible to alterations following prolonged VX exposure, and determine for the first time the threshold sub-acute VX dose for minimal physiological effects (up to 20% reduction in ChE activity) in the rat as 0.225 µg/kg/day.
Asunto(s)
Inhibidores de la Colinesterasa/toxicidad , Agentes Nerviosos/toxicidad , Compuestos Organotiofosforados/toxicidad , Animales , Biomarcadores/sangre , Temperatura Corporal , Peso Corporal , Encéfalo/efectos de los fármacos , Proteínas Portadoras/metabolismo , Sustancias para la Guerra Química/toxicidad , Colinesterasas/sangre , Colinesterasas/metabolismo , Relación Dosis-Respuesta a Droga , Masculino , Actividad Motora , Músculos/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M2/metabolismo , Receptores de GABA-A/metabolismo , Pruebas de Toxicidad CrónicaRESUMEN
Here we provide the first evidence that tetraspanin CD151 can support de novo carcinogenesis. During two-stage mouse skin chemical carcinogenesis, CD151 reduces tumor lag time and increases incidence, multiplicity, size and progression to malignant squamous cell carcinoma (SCC), while supporting both cell survival during tumor initiation and cell proliferation during the promotion phase. In human skin SCC, CD151 expression is selectively elevated compared with other skin cancer types. CD151 support of keratinocyte survival and proliferation may depend on activation of transcription factor STAT3 (signal transducers and activators of transcription), a regulator of cell proliferation and apoptosis. CD151 also supports protein kinase C (PKC)α-α6ß4 integrin association and PKC-dependent ß4 S1424 phosphorylation, while regulating α6ß4 distribution. CD151-PKCα effects on integrin ß4 phosphorylation and subcellular localization are consistent with epithelial disruption to a less polarized, more invasive state. CD151 ablation, while minimally affecting normal cell and normal mouse functions, markedly sensitized mouse skin and epidermoid cells to chemicals/drugs including 7,12-dimethylbenz[α]anthracene (mutagen) and camptothecin (topoisomerase inhibitor), as well as to agents targeting epidermal growth factor receptor, PKC, Jak2/Tyk2 and STAT3. Hence, CD151 'co-targeting' may be therapeutically beneficial. These findings not only support CD151 as a potential tumor target, but also should apply to other cancers utilizing CD151/laminin-binding integrin complexes.
Asunto(s)
Carcinoma de Células Escamosas/patología , Transformación Celular Neoplásica/patología , Integrina alfa6beta4/metabolismo , Neoplasias Cutáneas/patología , Tetraspanina 24/metabolismo , Tetraspanina 24/fisiología , 9,10-Dimetil-1,2-benzantraceno/toxicidad , Animales , Apoptosis , Western Blotting , Carcinógenos/toxicidad , Carcinoma de Células Escamosas/inducido químicamente , Carcinoma de Células Escamosas/metabolismo , Proliferación Celular , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Ratones , Ratones Noqueados , Fosforilación , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Neoplasias Cutáneas/inducido químicamente , Neoplasias Cutáneas/metabolismo , Células Tumorales CultivadasRESUMEN
The main injuries among victims of the terrorist act in the Tokyo subway resulted from sub-lethal inhalation and whole body exposure to sarin vapor. In order to study the long term effects of such exposure and to simulate these conditions, freely moving rats were exposed to sarin vapor (27.2±1.7 µg/l) for 10 min. About 50% of the rats showed no overt symptoms and the rest had mild to moderate clinical symptoms that subsided within 4h following exposure. A reduction of weight was noted during the first 3 days with full recovery on the 4th day. Rat's heart was challenged with epinephrine 1 and 6 months post exposure. A significant reduction in the threshold for epinephrine-induced arrhythmia (EPIA) was noted in rats exposed to sarin. A time dependent increase in the kD and Bmax values of muscarinic auto receptors (M2) was recorded in the rat's cortex and striatum. No changes were recorded in the rats' brain trans locator protein (TSPO) levels, concomitant with no observed changes in the animals' performance in A Morris water maze test. A significant increase in open field activity was noted 6 months following exposure to sarin vapor as well as a significant decrease in prostaglandin E2 (PGE2) production in the brain. It is speculated that down regulation of the M2 auto receptor function, caused hyper reactivity of the cholinergic system which leads to the changes described above. The continuous reduction in M2 auto-receptor system through an unknown mechanism may be the cause for long lasting decline in sarin-exposed casualties' health.
Asunto(s)
Encéfalo/efectos de los fármacos , Corazón/efectos de los fármacos , Exposición por Inhalación/efectos adversos , Sarín/administración & dosificación , Sarín/toxicidad , Animales , Encéfalo/fisiopatología , Corazón/fisiopatología , Dosificación Letal Mediana , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Ratas , Ratas Sprague-Dawley , Factores de Tiempo , VolatilizaciónRESUMEN
Anticholinesterases (antiChEs) are increasingly used for treating patients with neurodegenerative diseases, but the dependence of their effects on the integrity of cholinergic functions has not yet been analyzed at the molecular level. Here, we report that manipulation of muscarinic neurotransmission confers drastic changes on antiChE responses in the rat brain. In the brains of naïve, un-stressed rats, the irreversible organophosphate antiChE, diisopropylfluorophosphonate (DFP) induced post-treatment accumulation of catalytically active G1 monomers of acetylcholinesterase (AChE). Pre-treatment with the selective M1 muscarinic antagonist, pirenzepine, but not the general muscarinic antagonist, scopolamine, attenuated this G1 increase. DFP-enhanced AChE gene expression was accompanied by diverted splicing from the primary AChE-S mRNA variant, encoding G4 synaptic membrane AChE-S tetramers, to "readthrough" AChE-R mRNA, which encodes soluble G1 monomers. Both the mRNA increase and the shifted splicing were long lasting (>24 h) and common to the parietal cortex and hippocampal CA1 and CA3 neurons. Importantly, the splicing shift was maximal under DFP alone, as compared with sham-injected rats, and virtually preventable by pre-treatment with pirenzepine. In contrast, induction of AChE transcription was less dependent on muscarinic function, resulting in AChE-S but not AChE-R increases. Our findings demonstrate distinct regulation of the enhanced transcription and the alternative splicing reactions to antiChE treatment and shed new light on the differential responses to antiChEs of demented patients with increasingly impaired cholinergic neurotransmission.
Asunto(s)
Inhibidores de la Colinesterasa/farmacología , Antagonistas Muscarínicos/farmacología , Neuronas/efectos de los fármacos , Acetilcolinesterasa/genética , Acetilcolinesterasa/metabolismo , Animales , Isoflurofato/farmacología , Neuronas/enzimología , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Muscarínicos/metabolismo , Transcripción Genética/genéticaRESUMEN
Centrally mediated seizures and convulsions are common consequences of exposure to organophosphates (OPs). These seizures rapidly progress to status epilepticus (SE) and contribute to profound brain injury. Effective management of these seizures is critical for minimization of brain damage. Nasal application of midazolam (1.5 mg/kg) after 5 min of sarin-induced electrographic seizure activity (EGSA) ameliorated EGSA and convulsive behavior (238 +/- 90 s). Identical treatment after 30 min was not sufficient to ameliorate ECoG paradoxical activity and convulsive behavior. Nasal midazolam (1.5 mg/kg), together with scopolamine (1 mg/kg, im) after 5 min of EGSA, exerted a powerful and rapid anticonvulsant effect (53 +/- 10 s). Delaying the same treatment to 30 min of EGSA leads to attenuation of paroxysmal ECoG activity in all cases but total cessation of paroxysmal activity was not observed in most animals tested. Cognitive tests utilizing the Morris Water Maze demonstrated that nasal midazolam alone or together with scopolamine (im), administered after 5 min of convulsions, abolished the effect of sarin on learning. Both these treatments, when given after 30 min of convulsions, only decreased the sarin-induced learning impairments. Whereas rats which were not subject to the anticonvulsant agents did not show any memory for the platform location, both treatments (at 5 min as well as at 30 min) completely abolished the memory deficits. Both treatments equally blocked the impairment of reversal learning when given at 5 min. However, when administered after 30 min, midazolam alone reversed the impairments in reversal learning, while midazolam with scopolamine did not. Rats exposed to sarin and treated with the therapeutic regimen with the exclusion of midazolam exhibited severe brain lesions that encountered the hippocampus, pyriform cortex, and thalamus. Nasal midazolam at 5 min prevented brain damage, while delaying the midazolam treatment to 30 min of EGSA resulted in brain damage. The addition of scopolamine to midazolam did not alter the above observation. In summary, nasal midazolam treatment briefly after initiation of OP-induced seizure leads to cessation of EGSA and prevented brain lesions and behavioral deficiencies in the rat model.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Conducta Animal/efectos de los fármacos , Encéfalo/patología , Inhibidores de la Colinesterasa , Electroencefalografía/efectos de los fármacos , Hipnóticos y Sedantes/uso terapéutico , Midazolam/uso terapéutico , Sarín , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Administración Intranasal , Animales , Peso Corporal/efectos de los fármacos , Electrodos Implantados , Hipnóticos y Sedantes/administración & dosificación , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Midazolam/administración & dosificación , Antagonistas Muscarínicos/uso terapéutico , Ratas , Ratas Sprague-Dawley , Escopolamina/uso terapéutico , Convulsiones/psicología , Transferencia de Experiencia en Psicología/efectos de los fármacosRESUMEN
Epinephrine-induced arrhythmias (EPIA) are known to be associated with local cardiac cholinergic activation. The present study examined the development of QT prolongation and the effect on EPIA of whole-body exposure of animals to a potent acetylcholine esterase inhibitor. Freely moving rats were exposed to sarin vapor (34.2 +/- 0.8 microg/liter) for 10 min. The electrocardiograms (ECG) of exposed and control animals were monitored every 2 weeks for 6 months. One and six months post exposure, rats were challenged with epinephrine under anesthesia, and the threshold for arrhythmias was determined. Approximately 35% of the intoxicated rats died within 24 h of sarin exposure. Additional occasional deaths were recorded for up to 6 months (final mortality rate of 48%). Surviving rats showed, agitation, aggression, and weight loss compared to non-exposed rats, and about 20% of them experienced sporadic convulsions. Sarin-challenged rats with severe symptoms demonstrated QT segment prolongation during the first 2-3 weeks after exposure. The EPIA that appeared at a significantly lower blood pressure in the treated group in the first month after intoxication lasted for up to 6 months. This decrease in EPIA threshold was blocked by atropine and methyl-atropine. Three months post exposure no significant changes were detected in either k(D) or B(max) values of (3)H-N-methyl scopolamine binding to heart homogenates, or in the affinity of carbamylcholine to cardiac muscarinic receptors. The increase in the vulnerability to develop arrhythmias long after accidental or terror-related organophosphate (OP) intoxication, especially under challenging conditions such as stress or intensive physical exercise, may explain the delayed mortality observed following OP exposure.
Asunto(s)
Arritmias Cardíacas/inducido químicamente , Sustancias para la Guerra Química/toxicidad , Inhibidores de la Colinesterasa/toxicidad , Corazón/efectos de los fármacos , Sarín/toxicidad , Animales , Arritmias Cardíacas/fisiopatología , Atropina/farmacología , Conducta Animal/efectos de los fármacos , Presión Sanguínea/efectos de los fármacos , Inhibidores de la Colinesterasa/administración & dosificación , Electrocardiografía/efectos de los fármacos , Epinefrina , Exposición por Inhalación , Dosificación Letal Mediana , Síndrome de QT Prolongado/inducido químicamente , Síndrome de QT Prolongado/fisiopatología , Masculino , Antagonistas Muscarínicos/farmacología , Miocardio/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores Muscarínicos/efectos de los fármacos , Sarín/administración & dosificación , Escopolamina/metabolismo , Espectroscopía Infrarroja por Transformada de Fourier , VasoconstrictoresRESUMEN
The purpose of the present study was to compare the antidotal efficacy and the combined effects on inflammatory markers of three oximes--toxogonine, TMB4 and 2-PAM--in combination with anticholinergic drugs following exposure to sarin vapour by inhalation. Guinea pigs restrained in plethysmographs were exposed to various doses of sarin vapour (in the range of 1.4-4.4LD50). The antidotal mixture was injected immediately (5-20 s) following exposure (3 mg kg(-1) atropine and 1 mg kg(-1) benactyzine in combination with 6 mg kg(-1) toxogonine, 2 mg kg(-1) TMB4 or 12 mg kg(-1) 2-PAM). Bronchoalveolar lavage (BAL) samples were taken from surviving animals 24 h after exposure to determine the levels of inflammatory markers. A differential cell count was performed in BAL samples on Giemsa-stained slides. The inflammatory markers--histamine and prostaglandins (PGE)--were measured in BAL using radioimmunoassay (RIA) techniques. The survival rate in the various treatment groups and analysis of BAL samples showed that: (i) Toxogonine, TMB4 and 2-PAM, without pyridostigmine pretreatment, at doses that were proportional to their doses in the respective auto-injectors, exhibited similar antidotal efficacy against sarin exposure. (ii) The results demonstrated that a centrally acting anticholinergic drug is essential in the antidotal mixture to ensure survival. (iii) Histamine release and eosinophilia following sarin inhalation might require additional intervention, aimed at reducing the symptoms of allergic reaction and possibly expediting recovery.
Asunto(s)
Sustancias para la Guerra Química/toxicidad , Reactivadores de la Colinesterasa/farmacología , Exposición por Inhalación , Pulmón/inmunología , Pulmón/patología , Cloruro de Obidoxima/farmacología , Compuestos de Pralidoxima/farmacología , Compuestos de Piridinio/farmacología , Sarín/toxicidad , Animales , Biomarcadores/análisis , Lavado Broncoalveolar , Relación Dosis-Respuesta a Droga , Cobayas , Histamina/análisis , Inflamación , Pulmón/efectos de los fármacos , Masculino , Intoxicación/prevención & control , Prostaglandinas/análisis , Radioinmunoensayo , Sarín/administración & dosificación , VolatilizaciónRESUMEN
Chloroperoxidase (CPO) isolated from Caldariomyces fumago (20 U ml(-1)) together with urea hydrogenperoxide (UPER, 0.5 mM) and sodium chloride as co-substrate (NaCl, 0.5 M) caused rapid breakdown of VX (10 microM) (t((1/2)) = 8 s, 25 C, 50 mM tartarate, pH 2.75). Glucose oxidase (GOX, Aspergillus niger) and glucose were used as an alternative source for H(2)O(2). A mixture of GOX (20 U ml(-1)), glucose (GLU 0.45 M), CPO (20 U ml(-1)) and NaCl (0.5 M) caused a 3.8-fold slower degradation of VX (10 microM) (t((1/2)) = 30 s, 25 C, 50 mM tartarate, pH 2.75). The concentrations of H(2)O(2) and chlorine produced by this enzyme/substrate mixture depended mainly on the GLU concentration. Horseradish peroxidase (HRP) together with UPER (1 mM) and sodium iodide (NaI, 0.05 M) caused progressive degradation of VX that was more than 400-fold slower than with CPO (20 U ml(-1)), UPER (0.5 mM) and NaCl (0.5 M) (t((1/2)) = 55 min, 25 C, pH 8). Skin decontamination of VX by CPO was tested in pig-ear skin in vitro. The chemical agent VX (0.01 M, 100 microl) was degraded by 98% within 3 h of skin diffusion when a mixture of UPER/NaCl/CPO was applied 60 min prior to VX application. A mixture of UPER/NaCl without CPO also caused significant VX degradation (94%) during skin diffusion whereas it did not cause any VX degradation in solution. Degradation of VX in skin, obtained without exogenous CPO, may indicate involvement of endogenous intradermal haloperoxidase-like enzyme. Reagent UPER (1 mM) did not cause any degradation of VX in solution or during its skin diffusion. Furthermore, a mixture of CPO, UPER and NaCl caused rapid degradation of sulfur mustard (HD). Sulfur mustard (50 microM) incubated in the presence of CPO (4 U ml(-1)), UPER (0.05 M) and NaCl (0.5 M) at pH 2.75 and 30 C was oxidized by 97% and 99% within 5 and 10 min, respectively. The oxidation products HD sulfoxide, HD sulfone and HD sulfoxidevinyl were identified by GC/MS in the enzymatic chloroperoxidation mixture.
Asunto(s)
Sustancias para la Guerra Química/metabolismo , Cloruro Peroxidasa/metabolismo , Gas Mostaza/metabolismo , Compuestos Organotiofosforados/metabolismo , Peróxidos/metabolismo , Urea/metabolismo , Animales , Peróxido de Carbamida , Sustancias para la Guerra Química/análisis , Sustancias para la Guerra Química/toxicidad , Descontaminación/métodos , Cámaras de Difusión de Cultivos/métodos , Combinación de Medicamentos , Cromatografía de Gases y Espectrometría de Masas , Glucosa Oxidasa/metabolismo , Peroxidasa de Rábano Silvestre/metabolismo , Técnicas In Vitro , Masculino , Gas Mostaza/análisis , Compuestos Organotiofosforados/toxicidad , Piel/efectos de los fármacos , Piel/metabolismo , Porcinos , Urea/análogos & derivadosRESUMEN
Seizures and status epilepticus, which may contribute to brain injury, are common consequences of exposure to organophosphorus (OP) cholinesterase inhibitors. Effective management of these seizures is critical. To investigate the efficacy of nasal midazolam as an anticonvulsive treatment for OP exposure, as compared to intramuscular midazolam, guinea pigs were connected to a recording swivel for electrocorticograph (ECoG) monitoring and clinical observation. The experimental paradigm consisted of pyridostigmine pretreatment (0.1 mg/kg i.m.) 20 min prior to sarin exposure (1.2x LD(50,) 56 micro g/kg i.m.). One minute post-exposure, atropine (3 mg/kg i.m.) and TMB-4 (1 mg/kg im) were administered. Within 3-8 min after sarin exposure all animals developed electrographic seizure activity (EGSA), with convulsive behavior. Treatment with midazolam (1 mg/kg i.m.) 10 min after the onset of EGSA abolished EGSA within 389+/-181 s. The same dose was not effective, in most cases, when given 30 min after onset. However, a higher dose (2 mg/kg) was found efficacious after 30 min (949+/-466 s). In contrast, nasal application of midazolam (1 mg/kg) was found most effective, with significant advantages, in amelioration of EGSA and convulsive behavior, when given 10 min (216+/-185 s) or 30 min (308+/-122 s) following the onset of EGSA ( P<0.001). Thus, nasal midazolam could be used as a novel, rapid and convenient route of application against seizure activity induced by nerve agent poisoning.
Asunto(s)
Anticonvulsivantes/uso terapéutico , Inhibidores de la Colinesterasa/toxicidad , Agonistas del GABA/farmacología , Midazolam/uso terapéutico , Compuestos Organofosforados/toxicidad , Convulsiones/tratamiento farmacológico , Administración Intranasal , Animales , Anticonvulsivantes/administración & dosificación , Electrofisiología , Agonistas del GABA/administración & dosificación , Cobayas , Inyecciones Intramusculares , Midazolam/administración & dosificación , Sarín/toxicidad , Convulsiones/inducido químicamente , Convulsiones/fisiopatología , Factores de TiempoRESUMEN
The integrin alpha6beta4, a laminin receptor that stabilizes epithelial cell adhesion to the basement membrane (BM) through its association with cytokeratins, can stimulate the formation and stabilization of actin-rich protrusions in carcinoma cells. An important, unresolved issue, however, is whether this integrin can transmit forces to the substrate generated by the acto-myosin system. Using a traction-force detection assay, we detected forces exerted through alpha6beta4 on either laminin-1 or on an anti-alpha6 antibody, demonstrating that this integrin can transmit forces without the need to engage other integrins. These alpha6beta4-dependent traction forces were organized into a compression machine localized to the base of lamellae. We hypothesized that the compression forces generated by alpha6beta4 result in the remodeling of BMs because this integrin plays a major role in the interaction of epithelial and carcinoma cells with such structures. Indeed, we observed that carcinoma cells are able to remodel a reconstituted BM through alpha6beta4-mediated compression forces by a process that involves the packing of BM material under the cells and the mechanical removal of BM from adjacent areas. The distinct signaling functions of alpha6beta4, which activate phosphoinositide 3-OH kinase and RhoA, also contribute to remodeling. Importantly, we demonstrate remodeling of a native BM by epithelial cells and the involvement of alpha6beta4 in this remodeling. Our findings have important implications for the mechanism of both BM organization and tumor invasion.
Asunto(s)
Antígenos de Superficie/metabolismo , Membrana Basal/citología , Membrana Basal/metabolismo , Integrinas/metabolismo , Invasividad Neoplásica , Neoplasias/metabolismo , Neoplasias/patología , Membrana Basal/ultraestructura , Neoplasias de la Mama/metabolismo , Adhesión Celular , Matriz Extracelular/metabolismo , Humanos , Integrina alfa6beta4 , Laminina/metabolismo , Microscopía Electrónica , Microscopía Fluorescente , Microscopía por Video , Seudópodos/metabolismo , Transducción de Señal , Células Tumorales CultivadasRESUMEN
Although the involvement of alpha 6 beta 4, an integrin laminin receptor, in hemidesmosome organization has dominated the study of this integrin, recent studies are revealing novel functions for alpha 6 beta 4 in the migration of epithelial and carcinoma cells. The engagement of laminin by alpha 6 beta 4 can stabilize actin-rich protrusions and mediate traction forces necessary for cell movement. This integrin also has a significant impact on signaling molecules that stimulate migration and invasion, especially PI3-K and Rho GTPases. Activation of PI3-K by alpha 6 beta 4 enhances the formation of actin protrusions, and it may stimulate the function of other integrins, such as alpha 3 beta 1, that are also important for epithelial migration. Signaling through alpha 6 beta 4 may not always depend on the adhesive functions of this integrin, a possibility that has profound implications for migration and invasion because it implies that the ability of alpha 6 beta 4 to stimulate these processes is not limited to specific matrix environments.
Asunto(s)
Antígenos de Superficie/fisiología , Movimiento Celular , Células Epiteliales/fisiología , Integrinas/fisiología , Animales , Antígenos de Superficie/química , Carcinoma/patología , Adhesión Celular , Hemidesmosomas/metabolismo , Integrina alfa6beta4 , Integrinas/química , Modelos Biológicos , Invasividad Neoplásica , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
This review explores the mechanistic basis of breast carcinoma progression by focusing on the contribution of integrins. Integrins are essential for progression not only for their ability to mediate physical interactions with extracellular matrices but also for their ability to regulate signaling pathways that control actin dynamics and cell movement, as well as for growth and survival. Our comments center on the alpha6 integrins (alpha6beta1 and alpha6beta4), which are receptors for the laminin family of basement membrane components. Numerous studies have implicated these integrins in breast cancer progression and have provided a rationale for studying the mechanistic basis of their contribution to aggressive disease. Recent work by our group and others on mechanisms of breast carcinoma invasion and survival that are influenced by the alpha6 integrins are discussed.
Asunto(s)
Antígenos de Neoplasias/metabolismo , Antígenos de Superficie/metabolismo , Antígenos de Superficie/fisiología , Neoplasias de la Mama/metabolismo , Integrinas/fisiología , Neoplasias de la Mama/mortalidad , Movimiento Celular/fisiología , Progresión de la Enfermedad , Femenino , Humanos , Integrina alfa6beta4 , Fosfatidilinositol 3-Quinasas/metabolismo , Tasa de SupervivenciaRESUMEN
This report presents a non-lethal method for estimating a range of therapeutic doses of bisquaternary oximes that serve as antidotes against organophosphorus poisoning. We have estimated therapeutic oxime doses that are equivalent in their relative toxicity rather than selecting arbitrary fractions of their LD(50). Thus, toxic signs of the oximes HI-6, HLö-7, Toxogonin, AB-8 and AB-13 were monitored quantitatively in baboon monkeys and beagle dogs. Using Toxogonin as a reference oxime, a calculated unit of equivalent dose (CED) was defined as the oxime dose equal to the ratio between its minimal toxic dose (MTD) and the therapeutic ratio (TR) of Toxogonin i.e. CED = MTD/TR. Assuming that the tails of dose-response curves of toxicity for bisquaternary oximes are shallow and similar to one another, one could substitute the ED(10) for the MTD. The ED(10) values for bisquaternary oximes were estimated using the log-log model following experimental observations and quantitative scoring of toxic signs in dogs and monkeys. The MTD values then were calculated using the ED(10) values and the experimental therapeutic dose of the reference oxime Toxogonin. The following CED values were obtained for AB-8, AB-13, Toxogonin, HI-6 and HLö-7 in dogs (d) and monkeys (m): 98.7, 74.2, 30.0, 14.5 and 12.1 (d) and 281.9, 232.1, 41.7, 192.9 and 92.9 (m) micromol kg(-1), respectively. The antidotal efficacy of these oximes against poisoning by the nerve agent tabun was determined in dogs and monkeys. These dose-dependent efficacy data were obtained at 0.3 x CED, 1 x CED and 3 x CED of oximes in combination with atropine. These data provide comparative therapeutic values using oxime doses based on their relative toxicity. The highest antidotal efficacy against tabun in dogs was obtained for toxogonin, whereas HLö-7 and AB-13 were most efficacious in monkeys.
Asunto(s)
Cloruro de Obidoxima/uso terapéutico , Intoxicación por Organofosfatos , Oximas/uso terapéutico , Intoxicación/tratamiento farmacológico , Piridinas/uso terapéutico , Compuestos de Piridinio/uso terapéutico , Animales , Perros , Relación Dosis-Respuesta a Droga , Femenino , Masculino , Oximas/administración & dosificación , Oximas/toxicidad , Papio , Estadística como Asunto , Relación Estructura-Actividad , Sobrevida , Equivalencia Terapéutica , Factores de TiempoRESUMEN
The alpha(6) integrin is a 140-kDa (nonreduced) laminin receptor. We have identified a novel 70-kDa (nonreduced) form of the alpha(6) integrin called alpha(6)p for the latin word parvus, meaning small. The variant was immunoprecipitated from human cells using four different alpha(6)-specific monoclonal antibodies but not with alpha(3) or alpha(5) antibodies. The alpha(6)p integrin contained identical amino acid sequences within exons 13--25, corresponding to the extracellular "stalk region" and the cytoplasmic tail of the alpha(6) integrin. The light chains of alpha(6) and alpha(6)p were identical as judged by alpha(6)A-specific antibodies and electrophoretic properties. The alpha(6)p variant paired with either beta(1) or beta(4) subunits and was retained on the cell surface three times longer than alpha(6). Reverse transcription/polymerase chain reaction analysis revealed a single polymerase chain reaction product. The alpha(6)p variant was found in human prostate (DU145H, LnCaP, PC3) and colon (SW480) cancer cell lines but not in normal prostate (PrEC), breast cancer (MCF-7), or lung cancer (H69) cell lines or a variant of a prostate carcinoma cell line (PC3-N). Protein levels of alpha(6)p increased 3-fold during calcium-induced terminal differentiation in a normal mouse keratinocyte model system. A novel form of the alpha(6) integrin exists on cell surfaces that contains a dramatically altered extracellular domain.
Asunto(s)
Integrinas/análisis , Secuencia de Aminoácidos , Humanos , Integrinas/química , Integrinas/genética , Datos de Secuencia Molecular , Especificidad de ÓrganosRESUMEN
This article explores the mechanistic basis of carcinoma progression by focusing on the contribution of integrins. Integrins are essential for progression because of their ability to mediate physical interactions with extracellular matrices and their ability to regulate signaling pathways that control actin dynamics and cell movement, and for growth and survival. This article centers on a6 integrins (a6B1 and a6B4), which are receptors for the laminin family of basement membrane components. Numerous studies have implicated these integrins in cancer progression and have provided a rationale for studying the mechanistic basis of their contribution to aggressive disease.
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Integrinas/fisiología , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/fisiopatología , Actinas/fisiología , Antígenos de Superficie/fisiología , Movimiento Celular/fisiología , Citoesqueleto/fisiología , Progresión de la Enfermedad , Humanos , Integrina alfa6beta1 , Integrina alfa6beta4 , Transducción de Señal/fisiologíaRESUMEN
This review explores the mechanistic basis of carcinoma migration and invasion by focusing on the contribution of integrins. Integrins are essential for invasion not only for their ability to mediate physical interactions with extracellular matrices, but also for their ability to regulate signaling pathways that control actin dynamics and cell movement, as well as for growth and survival. Our comments center on a unique member of the integrin family, the alpha 6 beta 4 integrin, which is a receptor for the laminin family of basement membrane components. Numerous studies have implicated this integrin in the invasion of solid tumors and have provided a rationale for studying the mechanistic basis of its contribution to the invasive process. Such studies have revealed novel insights into the mechanism of carcinoma invasion that involve both the dynamics of cell migration and signaling pathways that regulate this migration.
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Antígenos de Neoplasias/metabolismo , Antígenos de Superficie/metabolismo , Integrinas/metabolismo , Invasividad Neoplásica , Neoplasias/metabolismo , Movimiento Celular , AMP Cíclico/metabolismo , Humanos , Integrina alfa6beta4 , Fosfatidilinositol 3-Quinasas/metabolismo , Transducción de SeñalRESUMEN
Superficial injury involving the mucosa of the gastrointestinal tract heals by a process termed restitution that involves epithelial sheet movement into the damaged area. The forces that drive epithelial sheet movement are only partially understood, although it is known to involve changes in the morphology of cells bordering the damage, such as the formation of large, flat, cytoplasmic extensions termed lamellae. We investigated the mechanism of epithelial sheet movement by following the response of the actin cytoskeleton and specific integrins (alpha6beta4, alpha6beta1, and alpha3beta1) to wounding. To model this event in vitro, monolayers of T84 cells, well-differentiated colon carcinoma cells, were damaged by aspiration and the ensuing response was analyzed by a combination of time-lapse video microscopy, fluorescence confocal microscopy and antibody inhibition assays. We show that wound healing begins with retraction of the monolayer. alpha6beta4 integrin is localized on the basal surface in structures referred to as type II hemidesmosomes that persist throughout this early stage. We hypothesize that these structures adhere to the substrate and function to retard retraction. Once retraction ceases, the wound is contracted initially by actin purse strings and then lamellae. Purse strings and lamellae produce a pulling force on surrounding cells, inducing them to flatten into the wound. In the case of lamellae, we detected actin suspension cables that appear to transduce this pulling force. As marginal cells produce lamellae, their basal type II hemidesmosomes disappear and the alpha6 integrins appear evenly distributed over lamellae surfaces. Antibodies directed against the alpha6 subunit inhibit lamellae formation, indicating that redistribution of the alpha6 integrins may contribute to the protrusion of these structures. Antibodies directed against the alpha3beta1 integrin also reduce the size and number of lamellae. This integrin's contribution to lamellae extension is most likely related to its localization at the leading edge of emerging protrusions. In summary, wounds in epithelial sheets initially retract, and then are contracted by first an actin purse string and then lamellae, both of which serve to pull the surrounding cells into the denuded area. The alpha6 integrins, particularly alpha6beta4, help contain retraction and both the alpha6 integrins and alpha3beta1 integrin contribute to lamellae formation.
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Actinas/metabolismo , Citoesqueleto/fisiología , Integrinas/metabolismo , Mucosa Intestinal/fisiología , Cicatrización de Heridas/fisiología , Anticuerpos Monoclonales/farmacología , Antígenos de Superficie/metabolismo , Movimiento Celular/fisiología , Células Epiteliales/patología , Células Epiteliales/fisiología , Humanos , Inmunoglobulina G/farmacología , Integrina alfa3beta1 , Integrina alfa6beta1 , Integrina alfa6beta4 , Integrinas/inmunología , Mucosa Intestinal/lesiones , Microscopía Confocal , Microscopía Fluorescente , Microscopía por Video , Receptores de Laminina/metabolismo , Células Tumorales Cultivadas , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Organophosphate poisoning is associated with adverse effects on the central nervous system such as seizure/convulsive activity and long term changes in neuronal networks. This study reports on investigations designed to assess the consequences of soman exposure on excitatory amino acids receptors in the rat brain. In addition, the protective effects of caramiphen which acts at these receptors, and scopolamine, which does not, was determined on soman-induced alteration in rat brain functions. Administration of soman (1xLD50) to pyridostigmine pretreated rats produced seizure activity (measured by EEG monitoring) in all animals tested. Estimation of [3H]MK-801 binding to brain membranes from intoxicated rats revealed a marked decrease in Bmax value 24 but not 2 hrs following soman administration. The specific nature of these effects of soman was demonstrated by the findings that [3H]flunitrazepam binding to central benzodiazepine receptors remained unchanged in soman-poisoned rat brain membranes. Both scopolamine and caramiphen, when used prophylactically prevented the lethal effect of soman and completely blocked the development of electrographic seizure activity (EGSA). In contrast, only caramiphen abolished soman-induced modifications in NMDA/ion channel characteristics. Caramiphen displaced [3H]MK-801 bound to the NMDA/ion channel complex, possibly by interacting with the Zn2+ site whereas scopolamine did not. Moreover, caramiphen, but not scopolamine, partially protected mice from NMDA-induced lethality. Thus, it is suggested that an important component of the protective efficacy of caramiphen against organophosphate poisoning might be attributed to its ability to modulate NMDA receptors in addition to its anticholinergic properties.
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Encéfalo/efectos de los fármacos , Ciclopentanos/farmacología , Receptores de N-Metil-D-Aspartato/fisiología , Escopolamina/farmacología , Soman/toxicidad , Animales , Encéfalo/metabolismo , Inhibidores de la Colinesterasa/toxicidad , Maleato de Dizocilpina/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Electroencefalografía/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Flunitrazepam/metabolismo , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos ICR , Parasimpatolíticos/farmacología , Unión Proteica , Bromuro de Piridostigmina/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Receptores de Glutamato/efectos de los fármacos , Convulsiones/inducido químicamente , Factores de TiempoRESUMEN
We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.