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Characteristics and regulation of proline transport in cultured glioblastoma cells.

ABSTRACT: L-Proline transport in C6 glioblastoma cells takes place mainly via a saturable Na(+)-dependent mechanism. The uptake process can be discriminated into two components, system A and system ASC. A minor proportion of L-proline transport is carried out by the ASC system, which appears to be constitutively expressed by the cell, but most is by system A which shows adaptive responses to amino acid deprivation and sensitivity to N-methyl-alpha-aminoisobutyric acid. The transport system is inhibited by proline derivatives, such as methyl and benzyl esters, and also hydroxyproline, and is stereospecific. Incubation of glioblastoma cells with phorbol 12-myristate 13-acetate led to concentration- and time-dependent decreases in L-proline transport. This effect could be mimicked by exogenous phospholipase C. Proline transport is significantly stimulated in the presence of Ca(2+)-mobilization agents and strongly inhibited in the absence of Ca2+. The present data suggest a complex regulation of L-proline transport by different kinases in glioblastoma cells.

SUBMITTER: Zafra F

PROVIDER: S-EPMC1137284 | biostudies-other | 1994 Sep

REPOSITORIES: biostudies-other

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Project description:AimTo study short dsRNA oligonucleotides (siRNA) as a potent tool for artificially modulating gene expression of N-Myc down regulated gene 1 (NDRG1) gene induced under different physiological conditions (Normoxia and hypoxia) modulating NDRG1 transcription, mRNA stability and translation.MethodsA cell line established from a patient with glioblastoma multiforme. Plasmid DNA for transfections was prepared with the Endofree Plasmid Maxi kit. From plates containing 5 × 10(7) cells, nuclear extracts were prepared according to previous protocols. The pSUPER-NDRG1 vectors were designed, two sequences were selected from the human NDRG1 cDNA (5'-GCATTATTGGCATGGGAAC-3' and 5'-ATGCAGAGTAACGTGGAAG-3'. reverse transcription polymerase chain reaction was performed using primers designed using published information on β-actin and hypoxia-inducible factor (HIF)-1α mRNA sequences in GenBank. NDRG1 mRNA and protein level expression results under different conditions of hypoxia or reoxygenation were compared to aerobic control conditions using the Mann-Whitney U test. Reoxygenation values were also compared to the NDRG1 levels after 24 h of hypoxia (P < 0.05 was considered significant).ResultssiRNA- and iodoacetate (IAA)-mediated downregulation of NDRG1 mRNA and protein expression in vitro in human glioblastoma cell lines showed a nearly complete inhibition of NDRG1 expression when compared to the results obtained due to the inhibitory role of glycolysis inhibitor IAA. Hypoxia responsive elements bound by nuclear HIF-1 in human glioblastoma cells in vitro under different oxygenation conditions and the clearly enhanced binding of nuclear extracts from glioblastoma cell samples exposed to extreme hypoxic conditions confirmed the HIF-1 Western blotting results.ConclusionNDRG1 represents an additional diagnostic marker for brain tumor detection, due to the role of hypoxia in regulating this gene, and it can represent a potential target for tumor treatment in human glioblastoma. The siRNA method can represent an elegant alternative to modulate the expression of the hypoxia induced NDRG1 gene and can help to monitor the development of the cancer disease treatment outcome through monitoring the expression of this gene in the patients undergoing the different therapeutic treatment alternatives available nowadays.

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Characteristics and regulation of proline transport in cultured glioblastoma cells.

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