Figure 1
Sequestration of β-catenin by GFP-tagged E-cadherin lead to up-regulation of CLU levels in colon carcinoma cells. Changes in CLU protein levels were investigated in response to over-expression of GFP tagged E-cadherin and GFP as control. (A) Immunofluorescence was used to show the effects of transient over-expression of GFP tagged E-cadherin, which binds β-catenin, in LS174T and HCT116 colon carcinoma cell lines. In the left panel it is shown that the GFP-cyt-E-cadherin fusion protein efficiently sequesters β-catenin from the nucleus whereupon Wnt signaling is abrogated. In transfected cells (white arrow heads) β-catenin has a perinuclear localization contrasting untransfected cells (white arrows) where it is uniformly distributed. Shown in the right panel is the up-regulation of cytoplasmic CLU protein which follows the abrogation of Wnt signaling induced by the GFP-cyt-E-cadherin fusion protein. CLU is up-regulated in GFP-cyt-E-cadherin transfected cells (yellow arrow heads) but not in untransfected cells nor in cells transfected with GFP alone. (B) Western blot with cell lysates from LS174T cells 24 hr after transient transfection with GFP-cyt-E-cadherin and GFP as a control. β-actin was used as a loading control. CLU protein levels are up-regulated and c-MYC protein levels are down-regulated as a consequence of over-expressing GFP-cyt-E-cadherin but not GFP alone. WB: western blot, ab: Antibody.
Figure 2
Evaluation of dnTCF-Wnt-model system: Expression of exogenous gene products and phenotypic change. Doxycycline-inducible dominant negative (dn) versions of TCF1 and TCF4 factors, the effectors of the Wnt pathway, were over-expressed in stably transfected LS174T derived cell lines. (A) Proliferation was halted in LS174T derived cell lines after induction of both dnTCFs. This was visualised by methyl violet staining of cell cultures after 5 days of induction. (B) Similar results were obtained by manually counting cells in a haemocytometer after cells had been cultured in the presence (+) or absence (-) of induction for 4 days. Data are presented as the mean ± standard deviation from 3 separate experiments. (C) Induction of exogenous gene products, dnTCFs, was detected by western blot with cell lysates from LS174T derived cell lines 0, 12, and 24 hr after induction. Control cells are LS174T cells without any dnTCF expression vector. (D) Both dnTCF1 and dnTCF4 abrogate β-catenin/TCF driven transcription of the Wnt-target gene, c-MYC, as analyzed by western blot with cell lysates from LS174T derived cell lines 0 and 12 hr after induction.
Figure 3
>Intra- and extracellular CLU protein levels are increased specifically in response to dnTCF1 over-expression. Changes in CLU protein levels were investigated in response to over-expression of dnTCF1 and dnTCF4 in stably transfected LS174T derived cell lines. (A) Western blot with cell lysates from LS174T derived cell lines 0, 12, and 24 hr after induction. β-actin was used as a loading control. CLU protein levels are up-regulated after induction of dnTCF1, but not dnTCF4, and appear as a strong 60 kDa band and a faint ~40 kDa band. (B) Immunofluorescence in LS174T dnTCF1 cells, fixed 24 hr after either induction (+dox) or no induction (-dox). Intracellular CLU levels are up-regulated in response to induction of dnTCF1. The subcellular localization of up-regulated CLU is mainly cytoplasmic (white arrows). No (primary) antibody control is also shown. (C) Over-expression of dnTCF1 in LS174T cells also led to increased levels of extracellular CLU as demonstrated by western blot with acetone-precipitated total proteins from culture supernatant 0, 25, and 48 hr after induction. CLU appears as a ~40 kDa smear which is consistent with the secreted form of CLU being a cleaved glycosylated protein that consists of two chains of similar molecular weight, held together by several disulfide bonds.
Figure 4
Over-expression of dnTCF1 lead to reduced viability and increased cell death rate in LS174T cells. Over-expression of dnTCF1 significantly increases the rate of cell death in LS174T cells 48 hr after induction (p < 0.005, student's t-test). This was determined using two cell death assays based on either trypan blue dye or DNA-intercalating dyes, propidium iodide (PI) and hoechst 342 (HST). LS174T control and dnTCF1 cell lines were cultured in the presence of induction. After 24 hr and 48 hr, floating and attached cells were stained with the respective dyes. Data from both assays are presented as the mean ± standard deviation from 3 separate experiments. (A) PI/HST assay: Cells were stained with PI (stains apoptotic/necrotic cells) and HST (stains viable cells). Percentage of PI stained cells relative to total cell number reflect the extent of cell death. (B) Trypan blue dye exclusion assay: Cells were stained with the trypan blue dye. Percentage of trypan blue stained cells (stains dead cells) relative to total cell number reflect extent of cell death. (C) MTT viability assay: The cells' capability to metabollically convert the MTT substrate was quantified, 48 hr after either induction (+dox) or no induction (-dox), by solubilizing MTT formazan crystals and performing spectrophotometry at 540 nm. Cell viability, which is proportional to the absorbance at 540 nm, decreases in dnTCF1 over-expressing LS174T cells relative to control cells.
Figure 6
Characterization of the 5'-UTR of the CLU34 transcript by 5'-end RACE and RT-PCR. A colon adenocarcinoma cDNA library was used for 5'-end rapid amplification of cDNA ends (RACE) to determine the sequence of the extreme 5'-end of CLU34 mRNA. None of the three cloned 5'-end RACE fragments or the RT PCR analysis supported the most 5' extreme end of the CLU34 transcript containing the putative TATA-box as suggested by the RefSeq database. The 5'-end RACE results were consistent with results from RT-PCR reactions which only yielded an amplification product if the sense primer was placed downstream of the putative TATA-box.
Figure 5
Genomic structure of CLU locus and exon-composition of CLU mRNA variants expressed in colonic cells. (A) Diagram depicts the genomic location of all CLU exons on chromosome 8 and potential TCF-binding sites (red arrows) predicted by the MatInspector software. (B) Exonic structure of CLU mRNA variants.
Figure 7
Alterations of CLU, p21, and c-MYC mRNA levels after induction of dnTCFs in LS174T cells. The expression levels of various transcripts were monitored by real time RT-PCR after induction of dnTCFs in LS174T derived cell lines. Expression levels were normalized to the Ubiquitin C (UBC) transcript. The CLU34 mRNA variant is specifically up-regulated in response to dnTCF1 over-expression in LS174T cells. Data are presented as the mean ± standard deviation from 2 separate experiments with each experiment consisting of the mean value of 3 independent determinations. c-MYC mRNA levels decrease, whereas p21CIP1/WAF1 mRNA levels increase after induction of both dnTCFs.
Figure 8
Expression kinetics of CLU mRNA and protein levels after induction of dnTCF1 in LS174T cells. Expression kinetics of CLU mRNA and protein levels were studied 0, 3, 6, and 12 hr after induction of dnTCF1 in LS714T cells. c-MYC and p21CIP1/WAF1 mRNA levels were also monitored. (A) Western blot with cell lysates from LS174T cells 0, 3, 6, and 12 hr after induction. CLU protein levels do not increase until 12 hr after induction. Exogenous dnTCF1 proteins are induced as early as 3 hr after induction, and c-MYC protein levels decrease in parallel. (B) Real time RT-PCR shows that CLU34 and p21CIP1/WAF1 mRNA levels do not increase until 12 hr after induction. c-MYC mRNA levels decrease 6 hr after induction. Data are presented as the mean ± standard deviation from 2 separate experiments with each experiment consisting of the mean value of 3 independent determinations.