Full-length Adenomatous Polyposis Coli Research Articles

Phosphorylation near nuclear localization signal regulates nuclear import of adenomatous polyposis coli protein.

Mutation of the adenomatous polyposis coli (APC) gene is an early step in the development of colorectal carcinomas. APC protein is located in both the cytoplasm and the nucleus. The objective of this study was to define the nuclear localization signals (NLSs) in APC protein. APC contains two potential NLSs comprising amino acids 1767-1772 (NLS1(APC)) and 2048-2053 (NLS2(APC)). Both APC NLSs are well conserved among human, mouse, rat, and fly. NLS1(APC) and NLS2(APC) each were sufficient to target the cytoplasmic protein beta-galactosidase to the nucleus. Mutational analysis of APC demonstrated that both NLSs were necessary for optimal nuclear import of full-length APC protein. Alignment of NLS2(APC) with the simian virus 40 large T antigen NLS (NLS(SV40 T-ag)) revealed sequence similarity extending to adjacent phosphorylation sites. Changing a serine residue (Ser(2054)) to aspartic acid mutated the potential protein kinase A site adjacent to NLS2(APC), resulting in both inhibition of the NLS2(APC)-mediated nuclear import of a chimeric beta-galactosidase fusion protein and a reduction of full-length APC nuclear localization. Our data provide evidence that control of APC's nuclear import through phosphorylation is a potential mechanism for regulating APC's nuclear activity.

Lack of Cyclooxygenase-2 Activity in HT-29 Human Colorectal Carcinoma Cells

Induction of cyclooxygenase-2 (COX-2) is an early event in the sequence of polyp formation to colon carcinogenesis. COX-2 is at elevated levels in human colorectal cancers and in tumors and polyps of mouse models of colorectal cancer. Mutation of the adenomatous polyposis coli (APC) gene is the initial event leading to colorectal cancer. Colorectal cells in culture which express mutant APC are often used to examine the association of COX-2 expression and apoptosis. The expression of full-length APC in HT-29 cells, a human colorectal carcinoma cell line which normally expresses truncated APC and highly expresses COX-2, inhibits cell growth through increased apoptosis and results in a down-regulation of COX-2 protein. In this report, we examine whether down-regulation of COX-2 is directly linked to the increase in apoptosis observed in these HT-29-APC cells. We present evidence that COX-2 and apoptosis are not linked since COX-2, although expressed, is catalytically inactive. Interestingly, the COX-2 cloned from HT-29 cells is catalytically active when transfected into HCT-116 cells, a colorectal cell line which normally does not express COX-2, but is not active in the HT-29 cell line itself.

Adenomatous polyposis coli (APC) protein moves along microtubules and concentrates at their growing ends in epithelial cells.

Adenomatous polyposis coli (APC) tumor suppressor protein has been shown to be localized near the distal ends of microtubules (MTs) at the edges of migrating cells. We expressed green fluorescent protein (GFP)-fusion proteins with full-length and deletion mutants of Xenopus APC in Xenopus epithelial cells, and observed their dynamic behavior in live cells. During cell spreading and wound healing, GFP-tagged full-length APC was concentrated as granules at the tip regions of cellular extensions. At higher magnification, APC appeared to move along MTs and concentrate as granules at the growing plus ends. When MTs began to shorten, the APC granules dropped off from the MT ends. Immunoelectron microscopy revealed that fuzzy structures surrounding MTs were the ultrastructural counterparts for these GFP signals. The COOH-terminal region of APC was targeted to the growing MT ends without forming granular aggregates, and abruptly disappeared when MTs began to shorten. The APC lacking the COOH-terminal region formed granular aggregates that moved along MTs toward their plus ends in an ATP-dependent manner. These findings indicated that APC is a unique MT-associated protein that moves along selected MTs and concentrates at their growing plus ends through their multiple functional domains.

Introduction of full-length APC modulates cyclooxygenase-2 expression in HT-29 human colorectal carcinoma cells at the translational level

Mutation of the adenomatous polyposis coli (APC) gene is associated with the earliest stages of colorectal tumorigenesis and appears to be responsible for the hereditary condition familial adenomatous polyposis (FAP). Evidence indicates that cyclooxygenase-2 (COX-2) is induced and at elevated levels in human colorectal cancers and in the polyps of mouse FAP models. We have used HT-29 cells, a human colorectal carcinoma cell line with a mutant carboxy-truncated APC gene, in which intact APC gene has been introduced under the control of an inducible promoter. These HT-29-APC cells provide a suitable model system to examine how COX-2 expression becomes dysregulated after loss of APC function. Induction of full-length APC causes the HT-29-APC cells to undergo apoptosis. However, differentiation, as measured by alkaline phosphatase activity, is not induced upon expression of full-length APC. Full-length APC protein has been shown to bind the intracellular protein beta-catenin and, as a result, the Lef/Tcf transcription factors are down-regulated. Analysis of APC immunoprecipitates demonstrate a time-dependent increase of beta-catenin interacting with full-length APC. Thus, the Lef/Tcf signaling pathway is intact at this point in these cells. Furthermore, upon expression of full-length APC, COX-2 protein expression is down-regulated while COX-2 mRNA levels remain the same. These data indicate that APC plays a role, either directly or indirectly, in the translational regulation of COX-2. Treatment of the HT-29-APC cells with sodium butyrate, an inducer of apoptosis, does not alter COX-2 protein expression. Thus, COX-2 down-regulation appears to be APC specific and not just due to apoptotic induction. APC appears to uniquely regulate COX-2 expression. The mechanism by which COX-2 protein expression is down-regulated in the HT-29-APC cells is under investigation.

Introduction of full-length APC modulates cyclooxygenase-2 expression in HT-29 human colorectal carcinoma cells at the translational level.

Mutation of the adenomatous polyposis coli (APC) gene is associated with the earliest stages of colorectal tumorigenesis and appears to be responsible for the hereditary condition familial adenomatous polyposis (FAP). Evidence indicates that cyclooxygenase-2 (COX-2) is induced and at elevated levels in human colorectal cancers and in the polyps of mouse FAP models. We have used HT-29 cells, a human colorectal carcinoma cell line with a mutant carboxy-truncated APC gene, in which intact APC gene has been introduced under the control of an inducible promoter. These HT-29-APC cells provide a suitable model system to examine how COX-2 expression becomes dysregulated after loss of APC function. Induction of full-length APC causes the HT-29-APC cells to undergo apoptosis. However, differentiation, as measured by alkaline phosphatase activity, is not induced upon expression of full-length APC. Full-length APC protein has been shown to bind the intracellular protein beta-catenin and, as a result, the Lef/Tcf transcription factors are down-regulated. Analysis of APC immunoprecipitates demonstrate a time-dependent increase of beta-catenin interacting with full-length APC. Thus, the Lef/Tcf signaling pathway is intact at this point in these cells. Furthermore, upon expression of full-length APC, COX-2 protein expression is down-regulated while COX-2 mRNA levels remain the same. These data indicate that APC plays a role, either directly or indirectly, in the translational regulation of COX-2. Treatment of the HT-29-APC cells with sodium butyrate, an inducer of apoptosis, does not alter COX-2 protein expression. Thus, COX-2 down-regulation appears to be APC specific and not just due to apoptotic induction. APC appears to uniquely regulate COX-2 expression. The mechanism by which COX-2 protein expression is down-regulated in the HT-29-APC cells is under investigation.

The Ubiquitin-Proteasome Pathway and Serine Kinase Activity Modulate Adenomatous Polyposis Coli Protein-mediated Regulation of β-Catenin-Lymphocyte Enhancer-binding Factor Signaling

The tumor suppressor function of the adenomatous polyposis coli protein (APC) depends, in part, on its ability to bind and regulate the multifunctional protein, beta-catenin. beta-Catenin binds the high mobility group box transcription factors, lymphocyte enhancer-binding factor (LEF) and T-cell factor, to directly regulate gene transcription. Using LEF reporter assays we find that APC-mediated down-regulation of beta-catenin-LEF signaling is reversed by proteasomal inhibitors in a dose-dependent manner. APC down-regulates signaling induced by wild type beta-catenin but not by the non-ubiquitinatable S37A mutant, beta-catenin. Bisindoylmaleimide-type protein kinase C inhibitors, which prevent beta-catenin ubiquitination, decrease the ability of APC to down-regulate beta-catenin-LEF signaling. All these effects on LEF signaling are paralleled by changes in beta-catenin protein levels. Lithium, an inhibitor of glycogen synthase kinase-3beta, does not alter the ability of APC to down-regulate beta-catenin protein and beta-catenin-LEF signaling in the colon cancer cells that were tested. These results point to a role for beta-catenin ubiquitination, proteasomal degradation, and potentially a serine kinase other than glycogen synthase kinase-3beta in the tumor-suppressive actions of APC.

Adenomatous Polyposis Coli Gene Mutation Alters Proliferation through its β-Catenin-Regulatory Function in Aggressive Fibromatosis (Desmoid Tumor)

Aggressive fibromatosis is a monoclonal proliferation of spindle (fibroblast-like) cells. A subset of lesions contain somatic truncating adenomatous polyposis coli (APC) gene mutations, and all of the lesions contain an elevated beta-catenin protein level. A major function of APC is to regulate beta-catenin protein level. Beta-catenin has a dual function in the cell: it is a member of the adherens junction, and it binds transcription factors in the tcf-lef family, transactivating transcription. Cell cultures from aggressive fibromatoses containing an APC mutation were studied. Transient transfection of the full-length APC gene caused decreased proliferation and beta-catenin protein level in these cultures. To determine whether beta-catenin protein level was responsible for the change in proliferation rate, stable transfections of deltaN89beta-catenin (a stabilized form that is not degraded by APC, but retains all other functions) were achieved in half of the cultures derived from each tumor, whereas the other half were transfected with an empty vector. Transfection of the full-length APC gene in cultures that were stably transfected with deltaN89beta-catenin did not result in a change in proliferation. The type I promotor of p56lck contains an HMG consensus region, to which members of the tcf-lef family can bind. p56lck was expressed in cultures not transfected with the full-length APC gene and in cultures transfected with the full-length APC gene and deltaN89beta-catenin, but not in cultures transfected with only the full-length APC gene. These data show that APC truncating mutations give aggressive fibromatosis cells a proliferative advantage through beta-catenin and suggest that beta-catenin acts to transactivate transcription.

Sequence-based design of a peptide probe for the APC tumor suppressor protein.

Proteins form specific associations, but predictive rules for protein pairing are generally unknown. Here, we describe amino-acid sequence patterns capable of mediating specific pairing of a widespread protein motif: the parallel, dimeric, alpha-helical coiled coil. The pairing rules were tested by designing a 54-residue peptide (anti-APCp1) that is predicted to dimerize preferentially with a coiled-coil sequence from the adenomatous polyposis coli (APC) tumor suppressor protein. As judged by circular dichroism, ultracentrifugation and native gel electrophoresis, anti-APCp1 formed a specific, helical, dimeric complex with the target APC coiled coil. On western blots of APC fragments expressed in Escherichia coli, the designed peptide detected a pattern of bands identical to the pattern detected by an antibody directed against the APC coiled coil. Peptide-mediated precipitation experiments showed that anti-APCp1 bound and sequestered wild-type and mutant APC proteins in extracts of human colon cancer cell lines. In addition, binding of the designed peptide preserved native APC-beta-catenin complexes. These biochemical experiments demonstrate that the anti-APC peptide preferentially forms a heterodimeric coiled coil with mutant and full-length APC proteins. The specificity of the designed peptide is sufficient to support several applications that commonly use antibodies. The observed specificity of anti-APCp1 validates the pairing rules used as the basis for the probe design, and it suggests that residues in the core positions of coiled coils help impart pairing selectivity.

Axin, a Negative Regulator of the Wnt Signaling Pathway, Directly Interacts with Adenomatous Polyposis Coli and Regulates the Stabilization of β-Catenin

The regulators of G protein signaling (RGS) domain of Axin, a negative regulator of the Wnt signaling pathway, made a complex with full-length adenomatous polyposis coli (APC) in COS, 293, and L cells but not with truncated APC in SW480 or DLD-1 cells. The RGS domain directly interacted with the region containing the 20-amino acid repeats but not with that containing the 15-amino acid repeats of APC, although both regions are known to bind to beta-catenin. In the region containing seven 20-amino acid repeats, the region containing the latter five repeats bound to the RGS domain of Axin. Axin and beta-catenin simultaneously interacted with APC. Furthermore, Axin stimulated the degradation of beta-catenin in COS cells. Taken together with our recent observations that Axin directly interacts with glycogen synthase kinase-3beta (GSK-3beta) and beta-catenin and that it promotes GSK-3beta-dependent phosphorylation of beta-catenin, these results suggest that Axin, APC, GSK-3beta, and beta-catenin make a tetrameric complex, resulting in the regulation of the stabilization of beta-catenin.

Nuclear and cytoplasmic localizations of the adenomatous polyposis coli protein.

Mutation of the adenomatous polyposis coli (APC) gene is an early step in the initiation of colon cancer. Because the distribution pattern of a protein within the cell can provide important clues as to function, we have used a combination of immunofluorescence microscopy and biochemical fractionation to determine the location of APC protein in epithelial cells. Immunofluorescence microscopy placed full-length APC protein in both the nucleus and the cytoplasm. The nuclear APC protein was concentrated in discrete subnuclear regions, including nucleoli, whereas the cytoplasmic APC protein concentrated at the leading edge of migrating cells. Colocalization of APC protein with rRNA confirmed a nucleolar localization. These immunocytochemical findings have been supported by cell fractionation, which demonstrated that full-length APC protein was located in both the membrane/cytoskeletal and the nuclear fractions.

Adenomatous polyposis coli tumor suppressor protein has signaling activity in Xenopus laevis embryos resulting in the induction of an ectopic dorsoanterior axis.

Mutations in the adenomatous polyposis coli (APC) tumor suppressor gene are linked to both familial and sporadic human colon cancer. So far, a clear biological function for the APC gene product has not been determined. We assayed the activity of APC in the early Xenopus embryo, which has been established as a good model for the analysis of the signaling activity of the APC-associated protein beta-catenin. When expressed in the future ventral side of a four-cell embryo, full-length APC induced a secondary dorsoanterior axis and the induction of the homeobox gene Siamois. This is similar to the phenotype previously observed for ectopic beta-catenin expression. In fact, axis induction by APC required the availability of cytosolic beta-catenin. These results indicate that APC has signaling activity in the early Xenopus embryo. Signaling activity resides in the central domain of the protein, a part of the molecule that is missing in most of the truncating APC mutations in colon cancer. Signaling by APC in Xenopus embryos is not accompanied by detectable changes in expression levels of beta-catenin, indicating that it has direct positive signaling activity in addition to its role in beta-catenin turnover. From these results we propose a model in which APC acts as part of the Wnt/beta-catenin signaling pathway, either upstream of, or in conjunction with, beta-catenin.

Expression of the APC gene after transfection into a colonic cancer cell line.

Mutations in the adenomatous polyposis coli (APC) gene cause the hereditary cancer syndrome familial adenomatous polyposis and are implicated in the early stages of sporadic colorectal carcinogenesis. APC is therefore a promising candidate for use in prophylactic gene therapy of intestinal tissues at high risk of becoming malignant. The aim of the study was to discover if functional full length APC gene can be introduced into somatic gut epithelial cells and to define the optimum conditions for such transfer. Copies of the normal APC gene were introduced into SW480 cells, a colonic epithelial cell line with an APC gene mutation, using plasmid DNA combined with liposomes. Reverse transcriptase polymerase chain reaction and restriction enzyme digestion allowed the endogenous gene to be distinguished from the transgene. It was shown that the normal APC gene is expressed at high levels for 72 hours after transfection and disappears within one week. This study shows that short-term expression of normal APC gene can be achieved after transfection with liposome-DNA complexes at sufficiently high levels to permit assessment of biological effects.

Purification of the Adenomatous Polyposis Coli (APC) Gene Product

Mutation of the adenomatous polyposis coli (APC) gene is responsible for familial adenomatous polyposis and is an etiologic factor for digestive tract malignancies. Although the APC gene product (APC) is believed to play a role in growth suppression of colonocytes, the underlying mechanism is not clear. However, recent evidence does suggest that APC is a microtubule-associated protein (MAP), and like other MAPs, it can be phosphorylated, as we have shown. To facilitate studies of APC function, we purified the APC protein. To purify the full-length APC protein, HCT116 human colon cancer cells were lysed and the particulate fraction from the lysate was extracted with ammonium sulfate followed by Sepharose 4B and DEAE-Sephacel column fractionation and then by sucrose zonal density gradient centrifugation. The final purified APC fraction was determined to be about 1000-fold enriched in APC. The availability of purified APC will be valuable in investigating possible growth-suppressing mechanisms of APC including specific sites of APC phosphorylation and APC's interaction with other cellular proteins.

Phosphorylation of the Adenomatous Polyposis Coli Protein and Its Possible Regulatory Effects in Cells

The adenomatous polyposis coli ( APC ) gene is etiologically associated with familial adenomatous polyposis and gastrointestinal malignancies, but its cellular function and role in tumorigenesis are unclear. Recent reports indicate that wild-type, but not mutant, APC gene product (APC) is associated with and promotes the assembly of cytoskeletal microtubules in vitro , suggesting that this mechanism has importance in tumor development. Because other microtubule-associated proteins (MAPs) undergo phosphorylation in their normal functioning, we postulated that APC is a phosphoprotein. HCT116 cells, containing full-length APC protein, were [ 32P]-prelabeled, and a 300-kDa band corresponding to phosphorylated APC was immunoprecipitated using each of three different anti-APC antibodies. High voltage electrophoresis of [ 32P]-labeled APC showed the presence of phospho-serine and phospho-threonine residues. Further immunoprecipitation analyses showed phosphorylation of i) full-length APC in human lymphoblastoid cells and ii) carboxyl-truncated APC in SW480 and DiFi colon carcinoma cells. Thus, APC is probably a phosphoprotein in normal and malignant tissues. We hypothesize a mechanism whereby phosphorylation of APC may play a regulatory role in its interaction with microtubules. This may involve phosphorylation of (Ser/Thr)-Pro amino acid motifs in APC′s basic domain. We propose that deletion of this domain disrupts APC binding to microtubules, explaining how APC mutations are linked to cancer development.

Loss of apc protein expressed by human colonic epithelial cells and the appearance of a specific low‐molecular‐weight form is associated with apoptosis IN vitro

APC (adenomatous polyposis coli) protein is differentially expressed in the normal colonic crypt and believed to be involved in colonic cell maturation. In this work we investigated whether expression of the APC protein is associated with cell death in colonic epithelial cells. We have previously reported an in vitro system to study apoptosis. Briefly, cells attached to the flask have a low frequency of apoptosis (1-3%), whereas cells that detach from the flask and float in the medium have a high proportion of apoptotic cells (36-96% depending on the cell line). The full-length 300-kDa or truncated APC protein, normally expressed by the attached cells (detected using the FE9 antibody), was found to be lost in the floating apoptotic cells in 8/11 colon tumour cell lines examined. In addition, the APC antibody FE9 detected a 90-kDa protein in the floating apoptotic cells of all cell lines investigated, which was not present in attached cells. Furthermore, loss of full-length APC and gain of the 90-kDa protein was observed in the apoptotic cells of 2 cell lines derived from other tissues: the SV40-transformed fibroblast cell line CMSV40fib and the lymphoblastoid B-cell line BJA-B. In cells repeatedly frozen and thawed, believed to induce necrotic cell death, full-length or truncated APC was also lost, though a 95-kDa protein distinct from that in apoptotic cells was observed. Specific loss of full-length or truncated APC (resulting in a 90-kDa protein in apoptotic cells but a 95-kDa protein in necrotic cells) is therefore associated with cell death. Our findings suggest a possible role for APC in cell survival.