RESUMEN
BAD, the proapoptotic member of the "BH3-only" subfamily of BCL-2 proteins, is inactivated by phosphorylation at serines 112 and 136 and by sequestration in the cytoplasm where it interacts with members of the 14-3-3 family. In BCR/ABL-expressing cells, BAD is constitutively phosphorylated and mainly cytoplasmic, whereas in cells expressing BCR/ABL mutants unable to protect from apoptosis, BAD is nonphosphorylated. We show here that both the wild-type (WT) and the S112A/ S136A double mutant (DM) BAD are more potent inducers of apoptosis in parental than in BCR/ABL-expressing 32D myeloid precursor cells. Stable lines of parental cells expressing DM BAD could not be established and most clones from WT BAD retrovirus-infected parental cells lost BAD expression. On IL-3 withdrawal from parental 32D cells, BAD was rapidly dephosphorylated by the serine-threonine phosphatase 1 alpha, and localized in the mitochondria, whereas it remained phosphorylated and did not localize to the mitochondria in the cohort of BCR/ABL-expressing cells escaping apoptosis induced by WT BAD. Moreover, these cells showed high levels of BCL-2 and BCL-X(L) expression. The cohort of BCR/ABL-expressing cells resistant to apoptosis induced by DM BAD showed only high levels of BCL-2 and BCL-X(L). These findings suggest that BCR/ABL-expressing cells are more versatile than normal hematopoietic progenitors in counteracting the apoptotic potential of BAD, and raise the possibility that tumor cells activate multiple antiapoptotic pathways for survival in the face of death-inducing stimuli. (Blood. 2000;96:676-684)
Asunto(s)
Apoptosis , Proteínas Portadoras/farmacología , Proteínas de Fusión bcr-abl/genética , Expresión Génica , Leucemia Mieloide/patología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Citoplasma/metabolismo , Células Madre Hematopoyéticas/metabolismo , Interleucina-3/administración & dosificación , Leucemia Mieloide/metabolismo , Ratones , Mitocondrias/metabolismo , Mutación , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Proteína Fosfatasa 1 , Proteína Letal Asociada a bclRESUMEN
Cell surface heparan sulfate proteoglycan (HSPG) interactions with type I collagen may be a ubiquitous cell adhesion mechanism. However, the HSPG binding sites on type I collagen are unknown. Previously we mapped heparin binding to the vicinity of the type I collagen N terminus by electron microscopy. The present study has identified type I collagen sequences used for heparin binding and endothelial cell-collagen interactions. Using affinity coelectrophoresis, we found heparin to bind as follows: to type I collagen with high affinity (Kd approximately 150 nM); triple-helical peptides (THPs) including the basic N-terminal sequence alpha1(I)87-92, KGHRGF, with intermediate affinities (Kd approximately 2 microM); and THPs including other collagenous sequences, or single-stranded sequences, negligibly (Kd >> 10 microM). Thus, heparin-type I collagen binding likely relies on an N-terminal basic triple-helical domain represented once within each monomer, and at multiple sites within fibrils. We next defined the features of type I collagen necessary for angiogenesis in a system in which type I collagen and heparin rapidly induce endothelial tube formation in vitro. When peptides, denatured or monomeric type I collagen, or type V collagen was substituted for type I collagen, no tubes formed. However, when peptides and type I collagen were tested together, only the most heparin-avid THPs inhibited tube formation, likely by influencing cell interactions with collagen-heparin complexes. Thus, induction of endothelial tube morphogenesis by type I collagen may depend upon its triple-helical and fibrillar conformations and on the N-terminal heparin-binding site identified here.
Asunto(s)
Colágeno/metabolismo , Endotelio Vascular/metabolismo , Heparina/metabolismo , Secuencia de Aminoácidos , Células Cultivadas , Colágeno/química , Humanos , Imitación Molecular , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Propiedades de SuperficieRESUMEN
Magnetic biodegradable poly(lactic acid) microspheres that incorporate both magnetite and the beta-emitter 90Y were prepared. By applying a directional external magnetic field gradient in excess of 0.02 Tesla/cm across a 96-well plate containing neuroblastoma cells incubated with the 90Y magnetite loaded microspheres, the radiation dose to the cells could be enhanced or reduced relative to the dose from a uniform loading of the well with 90Y-DTPA. Using the MTT assay, cell survival was measured for the magnetic field directed from above (cell sparing) and from below (cell targeting) the well plate, resulting in 65 +/- 8% or 18 +/- 5% survival respectively. This method was then applied to an in vivo murine tumor model. The biodistribution of intraperitoneally injected magnetic radioactive microspheres, after 24 h in mice, showed that 73 +/- 32% of the radioactivity was found on the subcutaneous tumor that had a rare earth magnet fixed above it. In contrast, the tumor radioactivity with no attached magnet was 6 +/- 4%. Magnetically targeted radiopolymers such as 90Y-microspheres show great promise for regional or intracavitary radiotherapy.
Asunto(s)
Supervivencia Celular/efectos de la radiación , Ácido Láctico , Linfoma/diagnóstico por imagen , Magnetismo , Ácido Pentético/administración & dosificación , Radioisótopos de Itrio/administración & dosificación , Animales , Línea Celular , Técnicas de Cultivo/métodos , Relación Dosis-Respuesta en la Radiación , Femenino , Humanos , Lactatos , Linfoma/metabolismo , Ratones , Ratones Endogámicos C57BL , Microesferas , Neuroblastoma , Ácido Pentético/farmacocinética , Poliésteres , Polímeros , Cintigrafía , Neoplasias Cutáneas/diagnóstico por imagen , Neoplasias Cutáneas/metabolismo , Distribución Tisular , Células Tumorales Cultivadas , Radioisótopos de Itrio/farmacocinéticaRESUMEN
High energy beta-emitting radioisotopes like Yttrium-90 have a radiotoxic range of about one centimeter. For cancer treatment they must be brought near the tumor cells and kept there for as long as they are radioactive. We developed as carriers for the ionic form of 90Y a matrix-type polymeric drug delivery system, poly(lactic acid) (PLA) microspheres. This radiopharmaceutical could be selectively delivered to the target site after incorporating 10% Fe3O4 (magnetite) which made the magnetic microspheres (MMS) responsive to an external magnetic field. Furthermore, MMS are biodegradable and slowly hydrolyze into physiologic lactic acid after the radioactivity is completely decayed. Previously prepared 10-40 microns MMS were radiochemically loaded to high specific activity with 90Y at a pH of 5.7. Stability studies showed that approximately 95% of added 90Y is retained within the PLA matrix after 28 days (> 10 half-lives) at 37 degrees C in serum, and electron microscopy showed that the microspheres retained their characteristic morphologic appearance for the same time period. Cytotoxicity studies with SK-N-SH neuroblastoma cells growing in monolayer showed that the radiocytotoxicity of the microspheres could be directed magnetically to either kill or spare specific cell populations, thus making them of great interest for targeted intracavitary tumor therapy. We are currently optimizing this system for use in the treatment of neoplastic meningitis.
Asunto(s)
Braquiterapia , Campos Electromagnéticos , Lactatos/química , Ácido Láctico , Polímeros/química , Radioisótopos de Itrio/administración & dosificación , Animales , Óxido Ferrosoférrico , Hierro , Ratones , Microscopía Electrónica de Rastreo , Microesferas , Neuroblastoma/radioterapia , Óxidos , Poliésteres , Propiedades de Superficie , Células Tumorales Cultivadas , Radioisótopos de Itrio/efectos adversosRESUMEN
Cerebrospinal fluid formation stems primarily from the transport of Na and Cl in choroid plexus (CP). To characterize properties and modulation of choroidal transporters, we tested diuretics and other agents for ability to alter ion transport in vitro. Adult Sprague-Dawley rats were the source of CPs preincubated with drug for 20 min and then transferred to cerebrospinal fluid (CSF) medium containing 22Na or 36Cl with [3H]mannitol (extracellular correction). Complete base-line curves were established for cellular uptake of Na and Cl at 37 degrees C. The half-maximal uptake occurred at 12 s, so it was used to assess drug effects on rate of transport (nmol Na or Cl/mg CP). Bumetanide (10(-5) and 10(-4) M) decreased uptake of Na and Cl with maximal inhibition (up to 45%) at 10(-5) M. Another cotransport inhibitor, furosemide (10(-4) M), reduced transport of Na by 25% and Cl by 33%. However, acetazolamide (10(-4) M) and atriopeptin III (10(-7) M) significantly lowered uptake of Na (but not Cl), suggesting effect(s) other than on cotransport. The disulfonic stilbene 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 10(-4) M), known to inhibit Cl-HCO3 exchange, substantially reduced the transport of 36Cl. Bumetanide plus DIDS (both 10(-4) M) caused additive inhibition of 90% of Cl uptake, which provides strong evidence for the existence of both cotransport and antiport Cl carriers. Overall, this in vitro analysis, uncomplicated by variables of blood flow and neural tone, indicates the presence in rat CP of the cotransport of Na and Cl in addition to the established Na-H and Cl-HCO3 exchangers.