ABSTRACT
Gliomas are the most common primary intracranial tumors. One extracellular matrix component that has been implicated in glial tumor biology is brain enriched hyaluronan binding (BEHAB)/brevican. In this study, the CNS-1 rat glioma cell line was transfected with a vector containing either a full-length BEHAB/brevican cDNA, a 5' insert encoding the NH(2)-terminal BEHAB/brevican cleavage product, or a 3' insert encoding the COOH-terminal cleavage product. As a control, CNS-1 cells were transfected with green fluorescent protein. Rats with intracranial grafts of BEHAB/brevican-transfected CNS-1 cells displayed significantly shorter survival times than did rats with CNS-green fluorescent protein intracranial grafts (P < 0.001). Histological examination showed that the BEHAB/brevican-transfected tumors were just as, if not more, aggressive than control tumors, even though the BEHAB/brevican tumors had been growing for only approximately two-thirds the time as long as control tumors. These data suggest that up-regulation and proteolytic cleavage of BEHAB/brevican increase significantly the aggressiveness of glial tumors. It will be important to investigate the effect of inhibiting cleavage of BEHAB/brevican in these cells and to determine the therapeutic potential of inhibiting BEHAB/brevican cleavage in gliomas.
Subject(s)
Brain Neoplasms/pathology , Carrier Proteins/physiology , Chondroitin Sulfate Proteoglycans/physiology , Glioma/pathology , Nerve Tissue Proteins/physiology , Animals , Brain Neoplasms/genetics , Brain Neoplasms/metabolism , Brevican , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Cell Division/genetics , Chondroitin Sulfate Proteoglycans/biosynthesis , Chondroitin Sulfate Proteoglycans/genetics , Glioma/genetics , Glioma/metabolism , Lectins, C-Type , Nerve Tissue Proteins/biosynthesis , Nerve Tissue Proteins/genetics , Rats , Rats, Inbred Lew , Transfection , Up-RegulationABSTRACT
BEHAB (Brain Enriched HyAluronan Binding)/brevican, a brain-specific member of the lectican family of chondroitin sulfate proteoglycans (CSPGs), may play a role in both brain development and human glioma. BEHAB/brevican has been cloned from bovine, mouse and rat. Two isoforms have been reported: a full-length isoform that is secreted into the extracellular matrix (ECM) and a shorter isoform with a sequence that predicts a glycophosphatidylinositol (GPI) anchor. Here, we report the characterization of BEHAB/brevican isoforms in human brain. First, BEHAB/brevican maps to human chromosome 1q31. Second, we report the sequence of both isoforms of human BEHAB/brevican. The deduced protein sequence of full-length, secreted human BEHAB/brevican is 89.7, 83.3 and 83.2% identical to bovine, mouse and rat homologues, respectively. Third, by RNase protection analysis (RPA) we show the developmental regulation of BEHAB/brevican isoforms in normal human cortex. The secreted isoform is highly expressed from birth through 8years of age and is downregulated by 20years of age to low levels that are maintained in the normal adult cortex. The GPI isoform is expressed at uniformly low levels throughout development. Fourth, we confirm and extend previous studies from our laboratory, here demonstrating the upregulation of BEHAB/brevican mRNA in human glioma quantitatively. RPA analysis shows that both isoforms are upregulated in glioma, showing an approximately sevenfold increase in expression over normal levels. In contrast to the developmental regulation of BEHAB/brevican, where only the secreted isoform is regulated, both isoforms are increased in parallel in human glioma. The distinct patterns of regulation of expression of the two isoforms suggest distinct mechanisms of regulation of BEHAB/brevican during development and in glioma.
Subject(s)
Cerebral Cortex/metabolism , Chondroitin Sulfate Proteoglycans/genetics , DNA, Complementary/genetics , Glioma/genetics , Nerve Tissue Proteins/genetics , Amino Acid Sequence , Base Sequence , Brain/metabolism , Brevican , Carrier Proteins/genetics , Cerebral Cortex/growth & development , Chondroitin Sulfate Proteoglycans/metabolism , Chromosome Mapping , Chromosomes, Human, Pair 1/genetics , Cloning, Molecular , DNA, Complementary/chemistry , Gene Expression , Gene Expression Regulation, Developmental , Gene Expression Regulation, Neoplastic , Humans , Lectins, C-Type , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , Proteoglycans/genetics , Proteoglycans/metabolism , RNA/genetics , RNA/metabolism , Transcription, GeneticABSTRACT
Overlapping cDNA clones spanning the entire coding region of a Na-channel alpha subunit were isolated from cultured Schwann cells from rabbits. The coding region predicts a polypeptide (Nas) of 1984 amino acids exhibiting several features characteristic of Na-channel alpha subunits isolated from other tissues. Sequence comparisons showed that the Nas alpha subunit resembles most the family of Na channels isolated from brain (approximately 80% amino acid identity) and is least similar (approximately 55% amino acid identity) to the atypical Na channel expressed in human heart and the partial rat cDNA, NaG. As for the brain II and III isoforms, two variants of Nas exist that appear to arise by alternative splicing. The results of reverse transcriptase-polymerase chain reaction experiments suggest that expression of Nas transcripts is restricted to cells in the peripheral and central nervous systems. Expression was detected in cultured Schwann cells, sciatic nerve, brain, and spinal cord but not in skeletal or cardiac muscle, liver, kidney, or lung.
Subject(s)
Schwann Cells/chemistry , Sodium Channels/genetics , Alternative Splicing , Animals , Base Sequence , Brain Chemistry , Cloning, Molecular , DNA Primers/chemistry , DNA, Complementary/genetics , Gene Expression , Genes , Molecular Sequence Data , Muscles/chemistry , Nerve Tissue Proteins/genetics , RNA, Messenger/genetics , Rabbits , Rats , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
A tetracycline-sensitive inducible expression system was used to regulate the expression of neurotransmitter receptor genes in two mammalian cell lines. The dopamine D3-receptor was stably expressed in GH3 cells, and GluR6 (a glutamate receptor subunit) was stably expressed in human embryonic kidney (HEK 293) cells. Three striking differences were found. 1) In the inactive state, virtually no D3-receptor expression was found in GH3 cells, whereas substantial levels of GluR6 expression were found in HEK 293 cells. 2) The induction of expression obtained upon removal of tetracycline was robust in GH3 cells but only modest in HEK 293 cells. 3) Whereas in each clonal cell line, the expression of a co-transfected hybrid transactivator is clearly regulated in a tetracycline-responsive manner, in the induced state, its mRNA levels were found to be very low in GH3 cells and very high in HEK 293 cells. The results indicate that, in contrast to GH3 cells, HEK 293 cells do not provide a cellular environment in which the expression of a heterologous gene can be tightly controlled in a tetracycline-responsive manner.
