Physical and functional characterization of the human LGI1 gene and its possible role in glioma development

Abstract

The human gene termed LGI1 (leucine-rich gene - glioma inactivated) has been isolated recently, and is supposed to be an additional candidate tumor suppressor gene involved in the formation and progression of glioblastoma multiforme [Chernova et al. (1998) Oncogene 17:2873-2881]. To test this hypothesis and to complete the characterization of the gene, we performed various detailed studies on the genomic structure, the mRNA expression level, the integrity of the cDNA, and retroviral gene transfer into LGI1-deficient cell lines. Two single nucleotide polymorphisms in the promotor region and a highly polymorphic intragenic microsatellite repeat between exon 4 and 5 were found. Phylogenetic sequence analysis techniques were applied, which showed functional relationships between LGI1 and TRK and SLIT protein families that are known to be involved in development and maintenance of the nervous system. Fluorescence in situ hybridization (FISH) analysis showed LGI1 to be present on 10q24 in each of 11 glioma-derived cell lines evaluated. Sequence analysis of the LGI1 transcript did not detect any mutation. Relative amounts of LGI1 mRNA copy numbers as measured by the real-time fluorescence detection LightCycler technology differed more than three orders of magnitude and were significantly reduced in 10 of 11 cell lines. Retroviral gene transfer into LGI1-deficient glioma-derived cell lines could not substantiate any difference to control infected cultures regarding growth rate, S phase transition, and maintenance of marker gene expression. The strong homology to proteins involved in development, differentiation, or maintenance of the nervous system provides evidence for a function of the LGI1 protein in neural tissue. The observation that translocation or deletion of the LGI1 locus or mutation of the coding sequence of the LGI1 mRNA is not a frequent event in malignant glioma cell lines suggests that epigenetic factors lead to substantial differences in the amount of LGI1 mRNA expression. In addition, that the effect is lacking after retroviral gene transfer in cell culture suggests that binding of some kind of a ligand is essential for its biological activity.

Suppression of the cell proliferation and invasion phenotypes in glioma cells by the LGI1 gene

“…In these experiments, the transfection efficiency appeared to be very low and the cells analysed were selected only for the presence of green fluorescence. Unlike the experiments we have presented here, where we were able to demonstrate the presence of the protein product in transfected cells using Western blots, Krex et al (2002) assumed that gene expression in the transfected cells was coincident with GFP. We have, however, frequently demonstrated that GFP constructs can dissociate and produce 'free' GFP in the cells.…”

“…A recent report by Krex et al (2002) failed to demonstrate any suppression of the transition through the S phase in glioma cell lines following introduction of a retroviral construct incorporating LGI1 and EGFP. In these experiments, the transfection efficiency appeared to be very low and the cells analysed were selected only for the presence of green fluorescence.…”

“…In addition, U87 cells were the only common cell line used between the two studies, which we clearly show is unaffected by expression of LGI1, presumably because of endogenous levels already present. The other cell lines used by Krex et al (2002), T1115, U373 and H4, were not included in our series but if they also express LGI1 it is unlikely that their malignant phenotype will be affected either. It is clear from the analysis of the FLAG6 clone from T98G that the levels of expression are important in response to the presence of LGI1 and so it is important to establish these levels before any comparison of data sets is possible.…”

LGI1mutations in autosomal dominant and sporadic lateral temporal epilepsy

“…However, neither point mutations affecting the LGI1 coding sequence nor differential methylation of its core promoter region could be demonstrated in these tumors, arguing against a role of LGI1 as a tumor suppressor gene [Krex et al, 2002;Piepoli et al, 2006;Somerville et al, 2000]. Recent studies have implicated LGI1 in the control of proliferation and invasiveness of glioma cell lines [Kunapuli et al, 2003].…”

The Tumor Suppressor Function of LGI1

“…Similar alterations occur also in the A172 glioma cell line and glioblastoma tumors. However, fluorescence in situ hybridization study showed that alterations of LGI1 gene are absent in some glioblastoma cell lines (Krex et al, 2002).…”