the leucine - rich , glioma inactivated 1 ( lgi1 ) gene was identified at the brake point of a reciprocal chromosome translocation t(10;19)(q24;q13 ) in the glioblastoma cell line t98 g .
lgi1 protein includes domains presumably involved in protein - protein interaction such as the leucine - rich repeats flanked by cysteine rich regions and the seven - bladed beta - propeller domain [ 24 ] .
lgi1 gene is predominantly expressed in brain , and it is associated with autosomal dominant lateral temporal epilepsy ( adlte ) [ 5 , 6 ] . although the specific function of lgi1 remains to be defined , it was suggested to form complexes with kv1.1 potassium channels , essential regulators of synaptic transmission in brain and also to be a ligand of the trans - membrane receptor adam22 , a protein highly expressed in brain whose function is still uncertain [ 7 , 8 ] .
the loss of lgi1 expression in most high - grade gliomas supported the function of tumor suppressor gene . in agreement with this hypothesis the reexpression of lgi1 in glioma cells , lacking endogenous lgi1 , impaired cell growth and migration capacity .
these phenotypic changes were linked with the downregulation of matrix metalloproteinase ( mmp ) genes by lgi1-mediated inhibition of the erk / mapk pathway . a role of lgi1 in the development of malignant brain tumors
was substantiated by the discovery of somatic missense mutations in lgi1 gene associated with high- and low - grade gliomas .
the ability of lgi1 to suppressor growth of neuroblastoma cells was suggested by the results of lgi1 overexpression , which was shown to impair proliferation and to induce apoptosis through the inhibition of the pi3k / akt pathway [ 12 , 13 ] .
further support to the tumor suppressor function of lgi1 was provided by a comprehensive study on malignant esophageal tumors ( barrett's - related adenocarcinomas ) showing that expression of lgi1 was consistently downregulated .
the aim of this study was to establish whether lgi1 was capable to suppress growth of cancer cells different from glioblastoma and neuroblastoma .
for this purpose we performed stable expression of lgi1 in hela cell , which is derived from a human cervical adenocarcinoma and expresses the human papillomavirus proteins e6/e7 ( hpv-18 ) .
essentially these viral proteins sustain cell proliferation and survival through the inactivation of tumor suppressors p53 and prb , which are implicated in cell cycle arrest and induction of apoptosis [ 16 , 17 ] . for these features conferring remarkably vigorous growth
the results showed that expression of lgi1 inhibited the rate of hela cell proliferation and increased cellular death .
furthermore lgi1 altered expression of key regulators of apoptosis such as the antiapoptotic b - cell lymphoma 2 gene ( bcl2 ) and of the proapoptotic b - cell lymphoma 2-associated x protein gene ( bax ) .
the lethal effect produced by lgi1 expression in hela cells suggests that the proposed role of tumor suppressor might be extended to adenocarcinoma - derived cells .
hela cells ( atcc number : ccl-2 ) were cultured in dulbecco 's modified eagle medium ( dmem , low glucose ) with 10% heat inactivated fetal calf serum ( fcs ) . for transfection cells ( 1.5  10 )
transfection with plasmid pclgi1 including the human lgi1 cdna ( 2075 bp ) or with empty vector pcdna3 ( 10 g / ml each ; invitrogen , groningen , the netherlands ) was performed by the dna ca - phosphate coprecipitation .
cells were cultured for 48 hours after transfection ; then they were replated at low density .
total rna was isolated from subconfluent cells by the geneelute mammalian total rna kit ( sigma chemical co. , st louis , mo , usa ) .
the cdna synthesis was performed by the reverse transcriptase improm - ii starting from random primer examer ( promega , madison , wi usa ) .
for quantification of lgi1 mrna , a cdna segment of ( 236 bp ) was amplified with primers : 5-tgtaaactgaaatggctagtggaa , and 5-agtaaaaggctgagcgatgactac .  
amplification of a bcl2 segment ( 258 bp ) was carried out with primers : 5-ggtcatgtgtgtggagagcgt , and 5-acttcacttgtggctcagataggc . a bax fragment ( 539 bp )
a portion of the glyceraldehyde-3-phosphate dehydrogenase ( gapdh , 909 bp ) was amplified using primers : 5-gaccccttcattgacctcaactaca and 5-ggtcttactccttggaggccatgt ( clontech , palo alto , ca usa ) . for the semiquantitative rt - pcr analysis
different number of pcr cycles ( < 25 ) and serial cdna dilutions were employed to establish conditions of linear amplification .
the pcr products were separated by agarose gel electrophoresis ; then the net intensity of ethidium bromide stained bands was determined using the kodak 1 d image analysis software .
the following antibodies ( santa cruz biotechnology , santa cruz , ca usa ) were used to probe blots : antibody c-19 directed to the c - terminal peptide of lgi1 ( 60 kda ) , affinity purified polyclonal antibody ( bax 21 ) against bax protein ( 23 kda ) , and monoclonal antibodies to bcl-2 and gapdh proteins ( 28 kda and 36 kda , respectively . the immunoreactions were revealed by the ecl reagent ( pharmacia amersham biotech , little chalfont , uk ) and quantified by the kodak 1 d image analysis software by evaluating the net intensity of the bands . an elisa immunoassay based on brdu incorporation was employed to measure cell proliferation according to the instruction procedures ( roches diagnostic , germany ) .
cells were plated in quadruplicate on 96 well cluster plates ( 100 cells / mm ) and cultured for 24 hours .
absorbance at ( 450690 nm ) was measured using a plate reader . to determine the fraction of dead cells , the lactate dehydrogenase ( ldh ) released in the medium and that present in intact cells was measured using the cytotox non - radioactive cytotoxicity assay ( promega , madison , wi usa ) .
cells were plated on 96-well plates ( 100 cells / mm ) and cultured for 24 hours .
one half of the culture medium was transferred to a fresh well to determine ldh spontaneously released by dead cells .
then the conversion of tetrazolium salt ( int ) into the red formazan product by ldh activity was measured ( absorbance 490 nm ) .
the fraction of dead cells was calculated as follows : % cytotoxicity = [ spontaneously released ldh  2  100]/total ldh . the apo - one homogeneous caspase-3/7 assay ( promega )
cells were seeded on 96-well plates ( 100 cells / mm ) , and the caspase-3/7 substrate z - devd - r110 was added 24 hours later .
the reaction was carried on for 6 hours , and the measurement of fluorescence was then performed by a plate spectrofluorimeter ( 485 nm excitation wavelength , 530 nm emission wavelength ) .
ldh assays were performed on replicated cell samples to determine the number of living cells , which was used to normalize caspase activity .
experimental data were expressed as the mean  sd . to determine the significance of variations , values were analyzed by student 's t - test ; levels of p < .05 were considered to be significant .
stable transfection of hela cells with lgi1 cdna was performed to establish whether expression of lgi1 might affect cell viability .
the level of lgi1 expression was evaluated in three cell clones designated he - lgi1 - 1 , he - lgi1 - 2 , and he - lgi1 - 3 , respectively ( figure 1 ) .
the results of the quantification of lgi1 mrna was quite in agreement with the estimated values of lgi1 protein ( table 1 ) .
although lgi1 protein was detected in whole cells sample , the possibility that it might be secreted as it occurred in 293 t cells   can not be excluded , since it might be bound to the cell surface .
the endogenous expression of lgi1 in nontransfected hela cells and in cell clones stably transfected with empty vector was undetectable by rt - pcr and western blotting ( see he - pcdna3 ; figure 1 and table 1 ) .
the absence of lgi1 expression in human uterine tumors and in normal uterine tissue is supported by the analysis of expressed sequence tags ( ests ) reported by unigene and is in agreement with lack of lgi1 expression in mouse uterus .
the brdu incorporation procedure was employed to evaluate the influence of lgi1 expression on the rate of hela cell proliferation .
all cell clones expressing lgi1 exhibited a lower proliferation rate in comparison with control cells transfected with empty vector ( figure 2 ) , which were similar to nontransfected hela cells ( not shown ) .
with he - lgi1 - 1 cell clone , in which lgi1 expression is very little , decrease of cell proliferation was on average 30% . with he - lgi1 - 2 cells , expressing the highest lgi1 levels the decrease was 80% , whereas it was 70% with helgi1 - 3 cells , showing intermediate levels of lgi1 expression ( figure 2 ) .
the results showed that the inhibitory effect of lgi1 on cell proliferation was proportional with the levels of lgi1 expression . to establish whether expression of lgi1 also might affect survival of hela cells we measured the amount of ldh spontaneously released in the medium as a consequence of cell death .
the fraction of dead cells was significantly greater in cell clones expressing lgi1 in comparison with control cells ( figure 3 ) .
in particular , the percentage of he - lgi1 - 1 dead cells was on average 7% , slightly but significantly greater than that of he - pcdna3 cells , which was 5% .
the lethal effect was much greater with he - lgi1 - 2 and he - lgi1 - 3 , whose fractions of dead cells were on average 26% and 21% , respectively .
the decline of cell survival was clearly correlated with the levels of lgi1 expression , likewise inhibition of cell proliferation . since
significant effects were obtained with cell clone helgi1 - 1 expressing very low levels of lgi1 , it can be suggested that the impairment of cell proliferation and survival was a consequence of the proper lgi1 activity rather than the outcome of artificial overexpression .
furthermore , these effects on cells of epithelial origin exclude the requirement of interactions with proteins specifically expressed in brain for the ability of lgi1 to contrast cell growth and survival . to gain information on the process of hela cell death caused by the expression of lgi1 we measured the activity of the apoptosis effectors caspase-3 and -7 in the experimental conditions employed to monitor cell death ( figure 4 ) .
the relative fluorescence emitted by the specific caspase-3/7 substrate upon cleavage was particularly enhanced with he - lgi1 - 2 ( 7.4-fold ) and he - lgi-3 cells ( 4.2-fold ) , whereas a modest increase was measured with he - lgi1 - 1 ( figure 3 ) .
the results supported the possibility that expression of lgi1 might trigger apoptosis of hela cells . to substantiate the induction of apoptosis of hela cell expressing lgi1 we measured the level of the antiapoptotic bcl2 and of the proapoptotic bax gene expression , since the relative proportion of these proteins plays a central role in the control of cell survival .
a significant decrease of bcl2 mrna and protein was measured in cell clones expressing lgi1 in comparison with control cells , whereas the expression of bax was consistently increased ( figure 1 ; table 1 ) . in particular , the upregulation of bax expression was proportional with the levels of lgi1 , with a maximum increase of 4.6-fold of bax protein in he - lgi1 - 2   ( table 1 ) .
the results showed that the antiapoptotic bcl2 gene was downregulated and that of its opponent proapoptotic bax was upregulated as a consequence of lgi1 expression .
a decreased ratio of bcl2/bax favors the release of apoptogenic molecules from mitochondria , which activate the intrinsic pathway of apoptosis in various cancer cells , including hela [ 2126 ] .
thus lgi1 might increase the susceptibility of hela cells to spontaneous apoptosis , similarly to what occurred in neurobalstoma cells .
apparently lgi1 was capable to counteract efficiently the oncogenicity of hpv , specifically of the viral protein e6 , which prevents apoptosis by stimulating the ubiquitin - mediated degradation of p53 , a transcriptional regulator capable to enhance the expression of bax and to repress that of bcl2 [ 16 , 2729 ] .
thus the balance of these apoptotic factors imposed by e6 to sustain cell survival might be destabilized by lgi1 in favor of apoptosis .
although the mechanism of cell death induced by lgi1 has not been completely elucidated , it seems possible that lgi1 might interfere with central survival pathways such as the pi3k / akt , as it occurred in neuroblastoma cells , because the prosurvival effects of this pathway includes transcriptional regulation of bcl2 and bax genes [ 30 , 31 ] . in conclusion
, this study supports the possibility that lgi1 might act as tumor suppressor of adenocarcinoma - derived cells , in line with the downregulation of lgi1 reported in barrett's - related adenocarcinomas and with previous studies on glioblastoma and neuroblastoma cells [ 914 ] .
 the lgi1 gene was suggested to function as tumor suppressor for its ability to reduce malignant features of glioblastoma cells . in support to this proposal were the findings that overexpression of lgi1 in neuroblastoma cells inhibited proliferation and induced apoptosis . in this study 
 we performed stable lgi1 expression in hela cells to examine whether the noxious effect of lgi1 might be extended to cancer cells of diverse origin . 
 hela cell clones stably expressing lgi1 exhibited a significant impairment of proliferation and a consistent increase of cell death when compared with control cells lacking expression of lgi1 . 
 expression of lgi1 increased the activity of apoptosis effectors caspase-3/7 ; furthermore it downregulated the antiapoptotic bcl2 gene and upregulated the proapoptotic bax gene expression , suggesting that the cause of hela cells death might be an increased susceptibility to apoptosis induced by lgi1 . 
 the results suggested that lgi1 is capable to restrain growth and survival of adenocarcinoma cells such as hela . 