ABSTRACT
Brain ischemia brings about hypoxic insults. Hypoxia is one of the major pathological factors inducing neuronal injury and central nervous system infection. We studied the involvement of mitogen-activated protein (MAP) kinase in hypoxia-induced apoptosis using cobalt chloride in C6 glioma cells. In vitro cytotoxicity of cobalt chloride was tested by MTT assay. Its IC(50) value was 400 microM. The DNA fragment became evident after incubation of the cells with 300 microM cobalt chloride for 24 h. We also evidenced nuclear cleavage with morphological changes of the cells undergoing apoptosis with electron microscopy. Next, we examined the signal pathway of cobalt chloride-induced apoptosis in C6 cells. The activation of extracellular signal-regulated protein kinase 1/2 (ERK 1/2) started to increase at 1 h and was activated further at 6 h after treatment of 400 M cobalt chloride. In addition, pretreatment of PD98059 inhibited cobalt chloride-induced apoptotic cell morphology in Electron Microscopy. These results suggest that cobalt chloride is able to induce the apoptotic activity in C6 glioma cells, and its apoptotic mechanism may be associated with signal transduction via MAP kinase (ERK 1/2).
Subject(s)
Apoptosis/drug effects , Cobalt/pharmacology , MAP Kinase Signaling System/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Line, Tumor , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Cell Size , DNA Fragmentation , DNA-Binding Proteins/metabolism , Enzyme Activation , Enzyme Inhibitors/metabolism , Flavonoids/metabolism , Glioma , Hypoxia/metabolism , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Mitogen-Activated Protein Kinase 3 , Nuclear Proteins/metabolism , Rats , Transcription Factors/metabolismABSTRACT
BACKGROUND/AIMS: Many genes encoding pituitary transcription factors involved in the formation of the pituitary gland are identified. Different mutations in these genes have been reported in patients with familial combined pituitary hormone deficiency (CPHD). This study was undertaken to analyze PIT1, PROP1, LHX3, and HESX1 in 12 CPHD patients with abnormal pituitary magnetic resonance imaging (MRI). Since embryonic development of the pituitary requires the coordinated expression of specific transcription factors, we postulated the presence of mutations in PIT1, PROP1, LHX3, and HESX1 genes. METHODS: Anterior pituitary function was evaluated. Each gene was PCR amplified exon by exon, and subsequently sequenced. RESULTS: In all cases, MRI examination showed abnormal pituitary gland development featuring ectopic neurohypophysis, hypoplastic anterior lobe, empty sella, and septo-optic dysplasia. Endocrinologically, all patients revealed multiple pituitary hormone deficiency including growth hormone, thyroid stimulating hormone, luteinizing hormone, follicular stimulating hormone and adrenocorticotropin. They were all sporadic cases without a positive family history. None of disease-causing specific mutations were identified in PIT1, PROP1, LHX3, and HESX1 genes of 12 sporadic CPHD patients with abnormal pituitary imaging. However, 2 novel polymorphisms were found in PROP1 gene: IVS1+3 A-->G and 27 T-->C (Ala9Ala) in exon 1. Their allele frequencies in patients and normal controls were not statistically different. Overall, allele frequencies of these polymorphisms were as follows: for the IVS1+3 A-->G polymorphism, the allele frequency of A was 54%, and 46% for G, with 58% of an A/G heterozygosity. For the 27 T-->C (Ala9Ala) polymorphism, the allele frequency of T was 46%, and 54% for G, with 42% of a T/C heterozygosity. CONCLUSIONS: Mutations of PIT1, PROP1, LHX3, and HESX1 genes are very rare in sporadic CPHD patients with abnormal pituitary MRI.
Subject(s)
Magnetic Resonance Imaging/methods , Pituitary Gland, Anterior/ultrastructure , Pituitary Gland, Posterior/ultrastructure , Pituitary Hormones/deficiency , Pituitary Hormones/genetics , Adolescent , Body Height/genetics , Child , Child, Preschool , Exons/genetics , Female , Gene Amplification , Gene Frequency , Humans , Infant , Male , Pituitary Gland, Anterior/pathology , Pituitary Gland, Anterior/physiopathology , Pituitary Gland, Posterior/abnormalities , Pituitary Gland, Posterior/pathology , Pituitary Hormones/blood , Polymerase Chain Reaction/methods , Polymorphism, Genetic , Sequence Analysis, DNA/methodsABSTRACT
Arsenic trioxide (As(2)O(3)) was recently demonstrated to be an effective inducer of apoptosis in patients with relapsed acute promyelocytic leukemia (APL) as well as in patients with APL in whom all-trans-retinoic acid and conventional chemotherapy failed. Chronic myelogenous leukemia cells are highly resistant to chemotherapeutic drugs. To determine if As(2)O(3) might be useful for the treatment of chronic myelogenous leukemia, we examined the ability of As(2)O(3) to induce apoptosis in K562 cells. In vitro cytotoxicity of As(2)O(3) was evaluated in K562 cells by a MTT assay; the IC(50) value for As(2)O(3) was determined to be 10 microM. When analyzed by agarose gel electrophoresis, the DNA fragments became evident after incubation of the cells with 20 microM As(2)O(3) for 24 h. We also found morphological changes and chromatin condensation of the cells undergoing apoptosis. Activation of caspase-3 was observed 6 h after treatment with 20 microM As(2)O(3) by a Western blot analysis. Next, we examined the MAP kinase-signaling pathway of As(2)O(3)-induced apoptosis in K562 cells. As(2)O(3) at 10 microM strongly induced the activation of p38 and JNK 1/2, while ERK 1/2 was inhibited. In addition, pretreatment of SB203580, a specific inhibitor of p38, inhibited As(2)O(3) induced apoptotic cell death. These results suggest that As(2)O(3) is able to induce the apoptotic activity in K562 cells, and its apoptotic mechanism may be associated with the activation of p38.
