The importance of -460 C/T and +405 G/C single nucleotide polymorphisms to the function of vascular endothelial growth factor A in colorectal cancer.

PURPOSE: The present study investigated the functional influence of the single nucleotide polymorphisms (SNPs) -460 C/T and +405 G/C at vascular endothelial growth factor A (VEGF-A), mRNA and protein levels in colorectal cancer (CRC) and normal colorectal tissue. METHODS: Blood and tissue were collected from 113 patients surgically resected for colorectal cancer. SNPs were analysed from genomic DNA by PCR, the VEGF-A gene expression analysis was performed by RT-PCR and protein analysis by ELISA. RESULTS: The T-allele in the -460 C/T SNP and the C-allele in the +405 G/C SNP were associated with significantly lower VEGF-A protein levels in normal colorectal tissue. There were no differences in protein levels in the malignant tissue according to genotypes. No differences were observed at the gene expression levels either. CONCLUSION: The results indicate that the two SNPs have a functional influence on the VEGF-A protein levels in normal colorectal tissue. The possible clinical implications of the findings need further investigation.

Quantitative analysis of vascular endothelial growth factor receptors 1 and 2 in colorectal cancer.

Vascular endothelial growth factor A (VEGF-A) is a key regulator of angiogenesis that binds to the receptors VEGFR-1 and VEGFR-2. It is well known that VEGF-A levels are increased in colorectal cancer (CRC) tissue compared to normal colorectal tissue, but little is known regarding the expression of the VEGFR-1 and -2 receptors. The aim of the present study was to perform a quantitative analysis of VEGFR-1 and -2 at the mRNA and protein level in tumour and normal colorectal tissue from CRC patients. Tissues were collected from 110 patients who underwent surgical resection for CRC. Receptor status was analysed at the gene expression level by real time RT-PCR using ß-2-microglobulin and ß-actin as reference genes. Protein analysis was performed using the ELISA technique. Gene expression and protein concentrations of VEGFR-1 and -2 were significantly increased in CRC tissue compared to normal colorectal tissue (P<10-6 for both receptors). An association between the gene expression of VEGFR-2 in CRC tissue and disease stage was detected (P=0.055). Significant correlations were also found between the gene expression of VEGFR-1 and -2 in CRC tissue and normal colorectal tissue (P<10-4). The gene expression and protein concentrations of VEGFR-1 and -2 were increased in CRC tissue compared to normal colorectal tissue, indicating that both receptors are important in CRC. The clinical implications of these findings require further investigation.

The transfer of heavy chains from bikunin proteins to hyaluronan requires both TSG-6 and HC2.

Tumor necrosis factor-stimulated gene-6 protein (TSG-6) is involved in the transfer of heavy chains (HCs) from inter-alpha-inhibitor (IalphaI), pre-alpha-inhibitor, and as shown here HC2.bikunin to hyaluronan through the formation of covalent HC.TSG-6 intermediates. In contrast to IalphaI and HC2.bikunin, pre-alpha-inhibitor does not form a covalent complex in vitro using purified proteins but needs the presence of another factor (Rugg, M. S., Willis, A. C., Mukhopadhyay, D., Hascall, V. C., Fries, E., Fülöp, C., Milner, C. M., and Day, A. J. (2005) J. Biol. Chem. 280, 25674-25686). In the present study we purified the required component from human plasma and identified it as HC2. Proteins containing HC2 including IalphaI, HC2.bikunin, and free HC2 promoted the formation of HC3.TSG-6 and subsequently HC3.hyaluronan complexes. HC1 or HC3 did not possess this activity. The presented data reveal that both HC2 and TSG-6 are required for the transesterification reactions to occur.

Molecular identification and characterisation of the glycine transporter (GLYT1) and the glutamine/glutamate transporter (ASCT2) in the rat lens.

Glutathione (GSH) is an essential antioxidant required for the maintenance of lens transparency. In the lens, GSH is maintained at unusually high concentrations as a result of direct GSH uptake and/or intracellular de novo synthesis from its precursor amino acids; cysteine, glycine and glutamine/glutamate. With increasing age, the levels of GSH, particularly in the core of the lens, are significantly reduced. It has been proposed that alterations in the transport of GSH and/or its precursor amino acids may contribute to the changes in GSH levels in older lenses. As considerable uncertainty exists about the molecular identity of GSH transporters in the lens, we have focused on identifying transporters involved in the uptake of the precursor amino acids required for GSH synthesis. Previously, we identified an uptake system for cyst(e)ine mediated by the Xc(-) exchanger and the Excitatory Amino Acid Transporters (EAATs) in the rat lens. In this current study, we have identified and localised additional uptake systems that contribute to GSH synthesis. Transcripts for GLYT1 (glycine transporter) and ASCT2 (glutamine/glutamate transporter) were detected in rat lens fiber cells using the reverse transcription-polymerase chain reaction (RT-PCR). Western blot analysis confirmed the expression of both GLYT1 and ASCT2 at the protein level. Immunocytochemistry revealed GLYT1 expression to be restricted to cortical regions of the lens. Labelling was predominantly cytoplasmic with some labelling of the membrane. In contrast, ASCT2 was expressed throughout the lens extending from the outer cortex through to the core. In the outer cortex, ASCT2 expression was predominantly cytoplasmic. However, with deeper distance into the lens, labelling became more membraneous indicating insertion of ASCT2 into the membranes of mature fiber cells of the lens core. The molecular identification and localisation of GLYT1 and ASCT2 in the lens suggests that these transporters may be responsible for the uptake of the precursor amino acids, glycine and glutamine, which are involved in GSH synthesis. Moreover, the presence of ASCT2 in the centre of the lens raises the possibility that ASCT2 may work with the Xc(-) exchanger to accumulate cysteine where it can potentially act as a low molecular mass antioxidant.