ABSTRACT
OBJECT: Cancer progenitor-like cells isolated by Hoechst 33342 dye efflux (termed the "side population" [SP]) have been studied in a variety of cancers, including malignant brain tumors. In this study, the authors investigate the nature of the SP phenotype in 2 glioma cell lines, U87MG and T98G, and their response to temozolomide. The roles of several adenosine triphosphate-binding cassette (ABC) multidrug transporters expressed by SP cells, in particular ABCG2, are also examined. METHODS: Using fluorescence-activated cell sorting, the cells were separated into SP and non-SP fractions and analyzed for progenitor cell-like properties with immunofluorescence staining, quantitative real-time polymerase chain reaction, and their ability to reform glioma mass in an immune-compromised mouse. The response of the SP cells to temozolomide was investigated at the cellular and molecular levels. Small interfering RNA knockdown was used to examine the specific role of the ABCG2 transporter, and the cells' tumorigenic potential was measured using the soft agar clonogenic assay. RESULTS: Side population cells are characterized by the presence of progenitor cell-like properties: increased expression of nestin, musashi-1, and ABCG2 were observed. In addition, only SP cells were able to reconstitute cellular heterogeneity; these cells were also more invasive than the non-SP cells, and possessed tumorigenic capacity. Temozolomide treatment increased the number of SP cells, and this corresponded to more progenitor-like cells, concurrent with elevated expression of several ABC transporters. CONCLUSIONS: Knockdown of ABCG2 transporters did not abrogate the SP cell response to temozolomide. Upregulation of several other ABC drug transporter genes is proposed to account for this chemoresistance.
Subject(s)
Antineoplastic Agents, Alkylating/pharmacology , Astrocytoma/pathology , Brain Neoplasms/pathology , Dacarbazine/analogs & derivatives , Glioma/pathology , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Animals , Antineoplastic Agents, Alkylating/therapeutic use , Astrocytoma/drug therapy , Astrocytoma/metabolism , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Dacarbazine/pharmacology , Dacarbazine/therapeutic use , Disease Models, Animal , Drug Resistance, Neoplasm , Gene Expression Regulation, Neoplastic/drug effects , Glioma/drug therapy , Glioma/metabolism , Humans , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/metabolism , Mice , Mice, Inbred BALB C , Mice, Nude , Neoplasm Invasiveness/pathology , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Nestin , RNA, Small Interfering/pharmacology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Temozolomide , Xenograft Model Antitumor AssaysABSTRACT
The gene mutated in the mouse open brain (opb) phenotype antagonizes sonic hedgehog-mediated signaling and encodes a small GTPase of the Rab family, Rab23. To date, the brain expression profile and exact mechanism of function of the Rab23 protein has remained unknown. Specific antibodies generated against Rab23 showed that the protein is highly enriched in the adult rodent brain and present in low levels in multiple tissues of the adult rodent. Rab23 is found in the cytosol as well as being associated with the plasma and endosomal membranes. In the adult mouse brain, Rab23 is found in betaIII tubulin (TuJ) positive neuronal cell bodies and are most prominent in the cortex, hypothalamus and the cerebellum. It is, however, absent from glial fibrillary acidic protein (GFAP) positive astrocytes or CNPase positive oligodendrocytes. Despite the plasma membrane/endosomal membrane localization of Rab23, neither overexpression of the GTP-restricted nor the GDP-bound mutant forms affect internalization of transferrin or epidermal growth factor. Exogenous overexpression of Rab23 or its mutants also did not affect the morphological differentiation of thalamic neurons in culture. Expression of Rab23 in the adult brain is suggestive, however, of having a postnatal function beyond its role in embryonic development.
Subject(s)
Brain/metabolism , Gene Expression Regulation, Developmental/physiology , Gene Expression/physiology , rab GTP-Binding Proteins/physiology , Age Factors , Animals , Blotting, Western/methods , Cells, Cultured , Embryo, Mammalian , Endocytosis/genetics , Humans , Immunohistochemistry/methods , Mice , Mutation/physiology , Neurites/physiology , Neuroglia/metabolism , Neurons/cytology , Subcellular Fractions/metabolism , Tissue Distribution/physiology , Transfection/methods , rab GTP-Binding Proteins/genetics , rab5 GTP-Binding Proteins/metabolismABSTRACT
To identify a major antigenic determinant for use in the development of a rapid serological diagnostic test for severe acute respiratory syndrome (SARS) coronavirus infection and to study the immune response during SARS coronavirus infection in humans, we cloned the full length and six truncated fragments of the nucleocapsid gene, expressed them, and purified them as glutathione S-transferase-tagged recombinant proteins. The reactivities of the recombinant proteins to a panel of antibodies containing 33 SARS coronavirus-positive sera and 66 negative sera and to antibodies against other animal coronaviruses were screened. A truncated 195-amino-acid fragment from the C terminus of the nucleocapsid protein (N195) was identified that had a strong ability to detect antibodies against SARS coronavirus. No cross-reaction was found between the N195 protein and antibodies against chicken, pig, and canine coronaviruses. The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples. The specificity and sensitivity of this test were 98.3 and 90.9%, respectively. The correlation between our Western blotting assay and an immunofluorescence assay (IFA) was also analyzed. The results of our Western blot assay and IFA for the detection of SARS coronavirus-positive sera were the same. Thus, the N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection.
