the wnt signaling pathway is known to play a key role in regulating cellular differentiation , proliferation , survival , and apoptosis [ 13 ] .
wnt proteins are a highly conserved family of secreted lipid - modified cysteine - rich glycoproteins   that activate signaling cascades inside the cell by binding to a member of the frizzled ( fz ) family of g - protein - coupled receptors on the extracellular matrix .
there are 19 members of vertebrate wnts , which can be grouped into two main classes based on their ability to stabilize cytosolic -catenin : canonical and noncanonical pathway [ 79 ] .
recent works have also shown that certain wnt ligands , like wnt5a , are able to activate more than only one wnt signaling pathway [ 10 , 11 ] , so the strictly binary classification is becoming more and more outdated . malfunctioning of wnt signaling pathway is related to several diseases like osteoporosis , crohn 's disease , alzheimer 's disease , schizophrenia , and especially cancer [ 1 , 16 ] .
the ligand wnt3a belongs to -catenin dependent ( canonical ) wnt signaling , which binds to frizzled transmembrane receptors .
subsequently the low - density lipoprotein receptor - related protein 5 ( lrp5 ) or lrp6 receptor complex activates cytoplasmic disheveled proteins , which trigger the inhibition of glycogen synthase kinase 3 ( gsk-3 ) .
the protooncoprotein -catenin accumulates in the cytoplasm as a consequence of the disassembly of the destruction complex that is formed by adenomatosis polyposis coli ( apc ) , axin , and gsk-3. thus , -catenin can be translocated into the nucleus where it activates the transcription of target genes mediated by t - cell specific transcription factor ( tcf)/lymphoid - enhancing factor ( lef ) .
these genes are responsible to regulate essential physiological processes in embryonic and adult development such as cell proliferation , differentiation , morphogenesis , and cell adhesion . with the indication of proliferation
, there is a complex interplay between the canonical wnt signaling and the cell cycle .
the wnt/-catenin signaling pathway is significant in stimulating progression of g1-phase by inhibiting gsk-3 , which regulates cell cycle effectors and growth regulators .
one direct target gene of the wnt/-catenin signaling pathway is the protooncogene myc , which was identified as a wnt target in colon cancer cells . the transcription regulator c
the cyclin d1 coding gene ccnd1 is also a target gene of the wnt/-catenin pathway , which was described in colon cancer cells [ 22 , 23 ] .
cyclin d1 as an activator of cyclin dependent kinase ( cdk ) 4 or cdk 6 drives the g1/s phase transition [ 24 , 25 ] .
one nonradioactive method is an immunoassay based on incorporation of 5-bromo-2-deoxyuridine ( brdu ) into dna to determine dna synthesis , which correlates to s phase transition and thus to cell proliferation .
assay an insoluble blue formazan product is produced by mitochondrial dehydrogenases by reduction of mtt ( 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide ) . in this study we investigated the proliferation of hek293 t cells under the influence of wnt3a ligands by using the mtt as well as the brdu assay .
furthermore we compare the results from these assays with mitochondrial activity of wnt3a treated cells as measured by immunostaining of the electron transport chain protein sdha ( succinate dehydrogenase complex subunit a ) .
the human embryonal kidney cells , hek293 t , were cultured in dulbecco 's modified eagle 's medium ( dmem ) supplemented with 10% fetal bovine serum ( pan biotech , aidenbach , germany ) , 1% nonessential amino acids , and 1% penicillin / streptomycin ( sigma aldrich , st .
ten thousand cells were seeded per well and cells were stimulated with escalating doses of wnt3a ( r&d systems , minneapolis , usa ) , namely , 0 , 10 , 50 , 100 , 150 , and 200  ng / ml , for 24 , 48 , and 72  h. mtt ( sigma - aldrich , st .
louis , usa ) was dissolved in pbs at 5  mg / ml and 20  l of mtt solution was added to each well followed by an incubation of three hours at 37c in 5% co2 .
medium with mtt was then flicked out and 200  l dmso was added . to dissolve the crystals ,
, mnnedorf , switzerland ) at 590  nm . for brdu assay a commercial kit
after addition of brdu labelling solution the cells were incubated for three hours at 37c in 5% co2 .
after the final washing step 100  l / well substrate solution was added to the cells and the reaction was stopped after 15  min incubation at room temperature by using sulphuric acid .
plates were read out immediately by an elisa reader at 450  nm . all assays were done in triplicate ( n = 12 ) . for calibration cells were seeded in different numbers ( 0,1  10 to 3  10 ) .
hek293 t cells were seeded into 24-well plates and treated with 0200  ng / ml wnt3a at a confluency of 30% and fixed in 4% formaldehyde after 24 , 48 , and 72  h. sdha antibody ( abcam , cambridge , uk ) at a dilution of 1  :  200 was added and cells were incubated for one hour .
z1 from zeiss , jena , germany ) and the accompanying software axiovision 4.7 . using the bradford - assay protein concentrations in whole - cell lysates were determined .
equal protein amounts were loaded on sds - polyacrylamide gels ( nupage , 412% , life technologies , carlsbad , usa ) .
gels were run at 150  v for 1  h and transferred onto nitrocellulose membranes via the iblot system ( life technologies ) .
membranes were analysed using primary antibodies against sdha , -catenin , c - myc , cyclin d1 , and -actin ( cell signaling , cambridge , uk ) , diluted 1  :  1000 in tbs - tween with 3% milk , followed by incubation with an appropriate horseradish peroxidase - conjugated secondary antibody ( dilution 1  :  2000 ) .
total rna was isolated using nucleospin rna ii ( macherey - nagel , dren , germany ) and 1  g rna was reverse - transcribed with verso cdna - kit ( thermo fisher scientific , schwerte , germany ) in a volume of 40  l .
sequences of the primers ( mwg , munich , germany ) used for semiquantitative reverse transcriptase pcr are for gapdh 5-catggtgctgagatttgccaac-3 ( forward ) and 5-tcaacaccttgaccttctcatcac-3 ( reverse ) , for myc 5-ccgagcaaggacgcgactctc-3 ( forward ) and 5-gcctttcagagaagcgggtcct-3 ( reverse ) , and for ccnd1 5-gcctgaacctgaggagcccca-3 ( forward ) and 5-gtcacacttgatcactctgg-3 ( reverse ) .
pcr amplification was performed using my taq red mix ( bioline , luckenwalde , germany ) according to the manufacturer 's protocol .
the reaction was performed with preliminary denaturation for 2  min at 94c , followed by 30 cycles of denaturation at 94c for 1  min , annealing at 55c ( ccnd1 ) , 57c ( gapdh ) , or 60c ( myc ) for 30  sec , and extension at 72c for 1  min .
western blots and agarose gels were scanned by gel documentation system quantum st4 ( vilber lourmat , eberhardzell , germany ) .
the bands were quantified by using imagej software ( wayne rasband , nih , bethesda , usa ) .
in order to prove activation of the wnt pathway , we detected the amount of -catenin protein concentration in hek293 t cells after treatment with wnt3a ( figure 1 ) .
after 24 hours of treatment , -catenin protein concentration rose with increasing concentrations of wnt3a .
the highest -catenin protein concentration was identified at 24 hours with a concentration of 200  ng / ml wnt3a . here
points 48 and 72 hours after treatment the effect of -catenin induction is not clearly recognizable any longer .
furthermore we analyzed the regulation of the wnt target genes ccnd1 and myc after treatment with wnt3a both by semiquantitative rt - pcr and by western blot ( figures 2 and 3 ) .
a basal level of c - myc and cyclin d1 was detected in untreated cells .
the results of rt - pcr showed marginal changes in the amount of myc cdna and an induction of ccnd1 after 24 and 48 hours with 10  ng / ml wnt3a , as well as after 72  h with a concentration of 100200  ng / ml wnt3a . a slightly increased protein level of c - myc was observed with a concentration of 50  ng / ml after 24 hours .
the western blot quantification of cyclin d1 showed an increase by a factor of 1,5 with a wnt3a concentration from 50 to 200  ng / ml after 24 hours .
the protein level of c - myc ascended in the same way with the concentration of 50  ng / ml wnt3a after 24 hours . to study the effects of wnt proteins on cell growth ,
hek293 t cells were grown in 96-well plates for 24 hours before they were treated with wnt3a protein concentrations of 0 , 10 , 50 , 100 , 150 , or 200  ng / ml , respectively .
after 24 , 48 , and 72 hours of incubation , mtt and brdu assays were performed .
the brdu assays show a clear trend ; with increasing concentrations of wnt3a the associated read - out increased ( figure 4(a ) ) , in which wnt3a indicates the highest growth rate after 48 hours and a concentration of 200  ng / ml .
in contrast to the brdu assays , the mtt assays did not show a correlation between wnt concentrations and cell numbers .
cell numbers stayed all about the same or decline in comparison to their relevant controls ( see figure 4(b ) ) . to further substantiate the role of wnt3a relating to the mitochondrial activity in hek293 t cells , amounts of sdha protein
succinate dehydrogenase ( sdh ) with its subunit sdha is a key component of the citric acid cycle , which is involved in complex ii of the mitochondrial electron transport chain and is responsible for transferring electrons from succinate to ubiquinone .
cells were treated equally to proliferation analysis experiments and incubated with sdha antibody as described above .
the results ( figure 5 ) showed an increasing fluorescence signal and also a more intense signal at western blot relative to the control after 24 hours of wnt3a treatment .
after 48 hours the protein level registered by immunofluorescence microscopy or by western blot analysis showed no significant change .
after 72 hours of wnt3a treatment even a decreasing signal was observed in both experiments .
the highest signal was measured after 24 hours with 50  ng / ml wnt3a .
wnt proteins are involved in various cellular functions including the regulation of gene expression for cell cycle and proliferation .
wnt3a is known as a promoter of proliferation and migration of human lens epithelial cells , also for proliferation of fibroblasts   or as a regulator of proliferation of pancreatic nit-1 beta cells .
exposed that wnt signaling activates mitochondrial biogenesis by performing a large - scale rnai screen ; they identified genes which affect mitochondrial function , amongst other myc . in this work we determined the effects of wnt3a on proliferation of hek293 t cells
for this we applied two major assays , which are widely used for analysis of proliferation rates of cultured cells , namely , the mtt and the brdu assays . in doing so , the specific effects of wnt ligands on dna synthesis and mitochondrial activity were detected and compared directly . in order to establish our system and to prove the effect of wnt3a on the wnt pathway in hek293 t cells we demonstrated the induction of -catenin after 24 hours of wnt3a treatment and we analyzed the expression levels of myc and ccnd1 .
c - myc has a key role in g1-phase progression ; it upregulates cyclin d1   and represses p21 and p27 [ 34 , 35 ] .
cyclin d1 in turn promotes phosphorylation and inhibition of the retinoblastoma ( rb ) complex , which itself upregulates the cyclin e level [ 19 , 36 ] .
the enrichment of cyclin e leads to the transition of the g1/s - phase checkpoint . during g1-phase mitochondria
this oxidative phase is followed by a reductive period during s-/g2-/m - phase of the cell cycle , in which replication of dna and proliferation of mitochondria take place .
proliferation rates of wnt3a treated hek293 t were different , when measured by the two assays used in this study .
the mtt assay measures the activity of mitochondria ; on the other hand the brdu assay detects the relative amount of newly synthesized dna .
refer to a highly significant relationship between brdu and mtt in their results with canine lymphocyte proliferation , although the brdu assay is proved to be more sensitive than the mtt assay .
the apparently negative effect of wnt3a on cell numbers could be explained by a decline in mitochondrial activity .
the brdu assay , however , showed a promoting effect on dna synthesis in these cells .
the experiments of mitochondrial activity showed that only after 24 hours of wnt3a treatment the sdha level was increased .
the activation with recombinant wnt3a has probably no effect on the mitochondrial activity after 48 hours or later .
according to this time - limited effect is the decreasing signal during mtt assay after 48 and 72 hours .
this is also confirmed by the declining accumulation of -catenin by wnt3a after 24 hours .
other publications support this assumption that wnt3a treated cells indicate an increased -catenin level only during a 24-hour period [ 41 , 42 ] .
we explain our results , which are contrary to the published proliferating effect of wnt3a by the fact that most studies used wnt3a overexpressed cells , wnt3a - conditioned medium , or serum - starved cells with recombinant wnt protein for activation wnt/-actin signaling pathway . in conclusion , results from brdu and the mtt assays are hardly comparable when measuring the effects of wnt3a on the proliferation rate of hek293 t cells .
differences can be explained by their different points of application , namely , the mitochondrial activity and the synthesis of dna . in order to give definitive evidence about the cellular proliferation rate , interpretation of outcomes of one single method might be misleading .
for evaluation of cell proliferation the direct counting of cells by optical methods would mirror best the real situation
. it would be interesting to compare the corresponding outcomes with the results of biochemical assays like brdu and mtt .
 the wnt signaling pathway has been associated with many essential cell processes . 
 this study aims to examine the effects of wnt signaling on proliferation of cultured hek293 t cells . 
 cells were incubated with wnt3a , and the activation of the wnt pathway was followed by analysis of the level of the -catenin protein and of the expression levels of the target genes myc and ccnd1 . 
 the level of -catenin protein increased up to fourfold . while the mrna levels of c - myc and cyclin d1 increased slightly , the protein levels increased up to a factor of 1.5 . 
 remarkably , mtt and brdu assays showed different results when measuring the proliferation rate of wnt3a stimulated hek293 t cells . in the brdu assays an increase of the proliferation rate 
 could be detected , which correlated to the applied wnt3a concentration . 
 oppositely , this correlation could not be shown in the mtt assays . 
 the mtt results , which are based on the mitochondrial activity , were confirmed by analysis of the succinate dehydrogenase complex by immunofluorescence and by western blotting . taken together , our study shows that wnt3a activates proliferation of hek293 cells . 
 these effects can be detected by measuring dna synthesis rather than by measuring changes of mitochondrial activity . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion