tretinoin and 3-methyladenine

Acute myeloid leukemia (AML) is characterized by the accumulation of immature blood cell precursors in the bone marrow. Pharmacologically overcoming the differentiation block in this condition is an attractive therapeutic avenue, which has achieved success only in a subtype of AML, acute promyelocytic leukemia (APL). Attempts to emulate this success in other AML subtypes have thus far been unsuccessful. Autophagy is a conserved protein degradation pathway with important roles in mammalian cell differentiation, particularly within the hematopoietic system. In the study described here, we investigated the functional importance of autophagy in APL cell differentiation. We found that autophagy is increased during all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of the APL cell line NB4 and that this is associated with increased expression of LC3II and GATE-16 proteins involved in autophagosome formation. Autophagy inhibition, using either drugs (chloroquine/3-methyladenine) or short-hairpin RNA targeting the essential autophagy gene ATG7, attenuates myeloid differentiation. Importantly, we found that enhancing autophagy promotes ATRA-induced granulocytic differentiation of an ATRA-resistant derivative of the non-APL AML HL60 cell line (HL60-Diff-R). These data support the development of strategies to stimulate autophagy as a novel approach to promote differentiation in AML.

Topics: Adaptor Proteins, Signal Transducing; Adenine; Antineoplastic Agents; Antirheumatic Agents; Autophagy; Autophagy-Related Protein 7; Autophagy-Related Protein 8 Family; Cell Differentiation; Chloroquine; Granulocytes; HL-60 Cells; Humans; Leukemia, Promyelocytic, Acute; Microfilament Proteins; Microtubule-Associated Proteins; Neoplasm Proteins; Tretinoin; Ubiquitin-Activating Enzymes

Increasing evidence suggests the toxicity of intracellular amyloid β-protein (Aβ) to neurons, as well as the involvement of oxidative stress in Alzheimer disease (AD). Here we show that normobaric hyperoxia (exposure of cells to 40% oxygen for five days), and consequent activation of macroautophagy and accumulation of Aβ within lysosomes, induced apoptosis in differentiated SH-SY5Y neuroblastoma cells. Cells under hyperoxia showed: (1) increased numbers of autophagic vacuoles that contained amyloid precursor protein (APP) as well as Aβ monomers and oligomers, (2) increased reactive oxygen species production, and (3) enhanced apoptosis. Oxidant-induced apoptosis positively correlated with cellular Aβ production, being the highest in cells that were stably transfected with APP Swedish KM670/671NL double mutation. Inhibition of γ-secretase, prior and/or in parallel to hyperoxia, suggested that the increase of lysosomal Aβ resulted mainly from its autophagic uptake, but also from APP processing within autophagic vacuoles. The oxidative stress-mediated effects were prevented by macroautophagy inhibition using 3-methyladenine or ATG5 downregulation. Our results suggest that upregulation of macroautophagy and resulting lysosomal Aβ accumulation are essential for oxidant-induced apoptosis in cultured neuroblastoma cells and provide additional support for the interactive role of oxidative stress and the lysosomal system in AD-related neurodegeneration.

Topics: Adenine; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Apoptosis; Autophagy; Autophagy-Related Protein 5; Cell Differentiation; Cell Nucleus; Cell Survival; Down-Regulation; Humans; Intracellular Space; Lysosomal Membrane Proteins; Lysosomal-Associated Membrane Protein 2; Lysosomes; Microtubule-Associated Proteins; Mutant Proteins; Neuroblastoma; Oxidants; Oxygen; Reactive Oxygen Species; RNA, Small Interfering; Transfection; Tretinoin; Tumor Cells, Cultured; Vacuoles

Cell differentiation is often associated with decreased cell growth, indicating an altered rate of macromolecule synthesis and degradation. In this study, we present evidence that autophagy, a process for bulk degradation of cytoplasm, is activated during retinoic acid-induced neuronal differentiation of neuroblastoma N2a cells. Chemical inhibitors of autophagy, including 3-MA and LY294002, abrogate cell differentiation. RNA interference of autophagy gene beclin 1 markedly delays the process of differentiation. We also find that cell differentiation is accompanied by decreased activity of mTOR, a major controller of cell growth and a negative regulator of autophagy. However, completely inhibiting mTOR by rapamycin decreases neurite outgrowth, cell size and the immunoreactivity for neuronal markers. Our study suggests that an appropriate level of mTOR activity is important in cell differentiation for a balance between macromolecule synthesis and degradation.

Topics: Adenine; Animals; Apoptosis Regulatory Proteins; Autophagy; Beclin-1; Carrier Proteins; Cell Differentiation; Cell Line, Tumor; Cell Size; Chromones; Dose-Response Relationship, Drug; Down-Regulation; Mice; Morpholines; Neurites; Neuroblastoma; Neurons; Phosphotransferases (Alcohol Group Acceptor); Protein Kinases; Proteins; Proto-Oncogene Proteins c-akt; RNA Interference; RNA, Small Interfering; Signal Transduction; Sirolimus; Time Factors; TOR Serine-Threonine Kinases; Tretinoin