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Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/β-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/β-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and β-catenin expression and BCL9-β-catenin co-localization. In addition, BCL9 formed a complex with β-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/β-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/β-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury. [ABSTRACT FROM AUTHOR]

Wnt signaling plays an important role in embryonic development and tumorigenesis. These biological effects are exerted by activation of theß-catenin/TCF transcription complex and consequent regulation of a set of downstream genes. TCF-binding elements have been found in the promoter regions of many TCF target genes and characterized by a highly conserved consensus sequence. Utilizing this consensus sequence, we performed an in silico screening for new TCF target genes. Through computational screening and subsequent experimental analysis, we identified a novel TCF target gene, DKK1, which has been shown to be a potent inhibitor of Wnt signaling. Our finding suggests the existence of a novel feedback loop in Wnt signaling.Oncogene (2004) 23, 8520-8526. doi:10.1038/sj.onc.1207892 Published online 20 September 2004 [ABSTRACT FROM AUTHOR]

The tumour suppressor adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC binds to β-catenin, a key component of the Wnt signalling pathway, and induces its degradation. APC interacts with microtubules and accumulates at their plus ends in membrane protrusions, and associates with the plasma membrane in an actin-dependent manner. In addition, APC interacts with the Rac-specific guanine nucleotide exchange factor Asef and stimulates its activity, thereby regulating the actin cytoskeletal network and cell morphology. Here we show that overexpression of Asef decreases E-cadherin-mediated cell-cell adhesion and promotes the migration of epithelial Madin-Darby canine kidney cells. Both of these activities are stimulated by truncated APC proteins expressed in colorectal tumour cells. Experiments based on RNA interference and dominant-negative mutants show that both Asef and mutated APC are required for the migration of colorectal tumour cells expressing truncated APC. These results suggest that the APC-Asef complex functions in cell migration as well as in E-cadherin-mediated cell-cell adhesion, and that truncated APC present in colorectal tumour cells contributes to their aberrant migratory properties. [ABSTRACT FROM AUTHOR]

The tumour suppressor gene adenomatous polyposis coli (APC) is mutated in sporadic and familial colorectal tumours. APC is involved in the proteasome-mediated degradation of β-catenin, through its interaction with β-catenin, GSK-3β and Axin. APC also interacts with the microtubule cytoskeleton and has been localized to clusters near the distal ends of microtubules at the edges of migrating epithelial cells. Moreover, in Xenopus laevis epithelial cells, APC has been shown to move along microtubules and accumulate at their growing plus ends. However, the mechanism of APC accumulation and the nature of these APC clusters remain unknown. We show here that APC interacts with the kinesin superfamily (KIF) 3A?KIF3B proteins, microtubule plus-end-directed motor proteins, through an association with the kinesin superfamily-associated protein 3 (KAP3). The interaction of APC with KAP3 was required for its accumulation in clusters, and mutant APCs derived from cancer cells were unable to accumulate efficiently in clusters. These results suggest that APC and β-catenin are transported along microtubules by KAP3?KIF3A?KIF3B, accumulate in the tips of membrane protrusions, and may thus regulate cell migration. [ABSTRACT FROM AUTHOR]

The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in many sporadic colorectal tumors. The carboxyl-terminal S/TXV motif of the APC gene product interacts with the PDZ domain of hDLG, the human homolog of the Drosophila lethal (1) discs large-1 (dlg) tumor suppressor. In the present study, we found that overexpression of hDLG suppresses cell proliferation by blocking cell cycle progression from the G0/G1 to S phase. This inhibition of cell cycle progression was abolished when the PDZ, SH3 or guanylate kinase-like domain of hDLG was mutated. Moreover, overexpression of these mutant hDLGs partially interfered with the cell cycle blocking activity of APC. Consistent with this result, mutant APC lacking the S/TXV motif exhibited weaker cell cycle blocking activity than the intact APC. These results suggest that APC-hDLG complex formation plays an important role in transducing the APC cell cycle blocking signal. Oncogene (2000) 19, 365–372. [ABSTRACT FROM AUTHOR]

APCL, a homologue of the adenomatous polyposis coli (APC) tumor suppressor, can deplete cytoplasmic beta-catenin like APC. However, as its biological function remains unclear, we have been using a yeast two-hybrid system to search for proteins that associate with its carboxyl region. Among several cDNA clones we isolated from a fetal-brain cDNA library as candidates, six included an identical sequence with significant homology to EB1, a protein known to bind to APC. The full-length cDNA of this novel homologue of EB1, named EB3, encoded a protein of 282 amino acids with 54% identity to EB1, and it was expressed preferentially in brain tissue on Northern blots. Confocal microscopy demonstrated that exogenous EB3, like EB1, is associated with the cytoplasmic microtubule network. Moreover, in these experiments EB3 and APCL appeared together in the perinucleus and the cytoplasmic microtubule network. Since APCL is also expressed highly and specifically in the central nervous system, APCL-EB3 interaction may be specific to the CNS, possibly involving stability and/or extension of microtubules during neuritogenesis.Oncogene (2000) 19, 210–216 [ABSTRACT FROM AUTHOR]

HTLV-1 Tax oncoprotein interacts with various cellular factors and modulates transcription and the cell cycle. To identify more cellular targets, we employed the yeast two hybrid system with Tax using a human cDNA library, and isolated a cDNA encoding the human counterpart of Drosophila discs large tumor suppressor protein, hDLG. Tax binding to hDLG was confirmed in vitro and also in HTLV-1-infected T-cells. Furthermore, hDLG was found to be efficiently phosphorylated in Tax-transfected cells and HTLV-1-infected T-cells. The C-terminus of Tax and the PDZ domain of hDLG were responsible for the binding of Tax to hDLG. The C-terminal peptide of Tax prevented the binding of hDLG to APC tumor suppressor gene product, suggesting inhibition of hDLG function by Tax. Over-expression of hDLG in NIH3T3 cells by microinjection induced a reduction of BrdU incorporation into DNA, but co-expression of Tax suppressed this inhibitory effect of hDLG. These results suggest that hDLG arrested the cell cycle and that Tax canceled this inhibitory action of hDLG through targeting hDLG. Therefore, Tax affects this novel regulatory pathway of the cell cycle alteration, of which seems to play a role in the development of human cancer. [ABSTRACT FROM AUTHOR]

The BCL-6 proto-oncogene encodes a 92- to 98-kDa transcriptional repressor containing the BTB/POZ domain at its N-terminal region and the zinc finger domain at its C-terminal region, respectively. In the present study, we examined the function of BCL-6 by using a recombinant adenovirus expressing BCL-6 (Ax1CA-BCL-6) and the lacZ reporter gene (Ax1CA-lacZ). Viability of CV-1 and HeLa cells infected with Ax1CA-BCL-6 was markedly reduced due to apoptosis, suggesting that BCL-6-overexpression induces apoptosis in CV-1 and HeLa cells. FACS analysis revealed that BCL-6-overexpressing cells are accumulated not only at the sub-G1 but also at G2/M phase. Induction of apoptosis by BCL-6 was preceded by down-regulation of apoptosis repressors BCL-2 and BCL-XL. These results suggest that BCL-6 induces apoptosis by regulating the expression of these apoptosis-regulating genes. [ABSTRACT FROM AUTHOR]

It has been suggested that the LGN protein is associated with Gαi2 by the yeast two-hybrid system and in vitro pull-down assay. To determine the functions of LGN in the central nervous system, we examined the expression and localization of LGN in mouse brain by immunoblotting and immunofluorescence microscopy. By immunoblotting, almost similar amounts of LGN were detected in the olfactory bulb, cerebral cortex, hippocampus, and cerebellum of the adult mouse brain, and the levels of the postnatal LGN expression in the whole brain were fairly constant. Immunofluorescence microscopy showed that LGN is localized in nuclei of the neurons in the olfactory bulb, cerebral cortex, and hippocampus, but in both nuclei and cytoplasm of Purkinje cells in the cerebellum. On the other hand, Gαi2 was distributed throughout the neuronal elements except for the nuclei. Thus, LGN and Gαi2 were colocalized in the cytoplasm of Purkinje cells, but not in other neurons examined. These results suggest that LGN may be involved not only in the Gαi2-mediated signaling but also in other signaling pathways. [ABSTRACT FROM AUTHOR]

Spa-1-like protein (SPAL) is a novel GTPase-activating protein (GAP) family protein. We generated anti-SPAL antibody, and examined localization of SPAL in the rat esophagus and heart by immunofluorescence microscopy. In the esophagus, SPAL was highly expressed in the spinous and granular layers and localized at the cell–cell border of the epithelial cells. SPAL in the spinous layer was almost colocalized with β-catenin, an intracellular anchor protein of E-cadherin, while colocalization of SPAL with the tight junction protein ZO-1 was shown in the granular layer. In the heart, SPAL was localized at the intercalated disks, and there colocalized with both β-catenin and ZO-1. In addition, much punctate localization of SPAL was seen along lateral borders of the cardiomyocytes, where β-catenin and ZO-1 were absent. From these results, it is suggested that SPAL may regulate cell–cell adhesion in the rat esophagus and heart. [ABSTRACT FROM AUTHOR]

In order to investigate the subnuclear interactions of the WTI gene product, nuclear fractionation analyses were performed with human osteosarcoma HOS and myelogenous leukemia K562 cells. The WT1 protein was tightly associated with the nucleus and was resistant to high-salt or derergent extraction and DNase I digestion. Both the expression level and stability of WT1 and its resistance to high salt and DNase I treatments remained constant during the cell cycle. In addition, human WT1 ectopically expressed in mouse NIH3T3 cells was also resistant to these treatments. These results suggest that WT1 functions in tight association with the nuclear matrix. [ABSTRACT FROM AUTHOR]

C-erbB- 2 is a human protooncogene homologous with the well-known c-erbB. Genes and gene products of the EGF receptor and c-erbB are known to be closely related and to be closely homologous in their intracellular domain. Inspection of the deduced amino acid sequence suggested that the c-erbB- 2 gene encodes a receptor for a yet unidentified growth factor. An immunohistological study was performed by introducing an antibody raised in the rabbit by immunization with a synthetic peptide corresponding to a part of the intracytoplasmic domain of predicted gene product. Specimens from 13 normal human organs, fresh frozen tissue from 41 surgically excised human malignant tumors and eight cell lines maintained in nude mice were studied. Positive staining was found in 4 of the 41 (9.8%) malignant tumors. All of the positive tumors were adenocarcinomas and two adenocarinoma cell lines were also positive. Amongst the normal human tissues, epithelial cells in stomach, small and large intestine were faintly stained. When the positively stained cell lines were studied by immunoelectronmicroscopy, the reaction was most prominent in the membrane of microvilli, but part of the nuclear membrane, the endoplasmic reticulum and the outer cell membrane were also stained. DNA and mRNA blot assays, as well as our immunoprecipitation test, revealed that immunohistologically positive cell lines bore amplified c-erbB- 2 DNA, c-erbB- 2 mRNA and 185 kD protein which is supposed to be the gene product, while negative cell lines did not. [ABSTRACT FROM AUTHOR]

The expression of midkine (MK), a growth/differentiation factor,was assessed in 34 surgically resected specimens ofprimary breast cancer or mastopathy. Using reverse transcriptase-polymerasechain reaction (RT-PCR) analysis, all of the non-cancerousand cancerous tissues were found to express MKexcept for one breast cancer specimen. Northern blotanalysis revealed that MK mRNA was also expressedin the normal breast tissues examined. Immunohistochemical analysisof the MK protein was performed on alimited number of the specimens, showing that somecancerous tissues were immunoreactive with anti-MK antibodies. Furthermore,using RT-PCR analysis, expression of not only thewild-type but also a truncated form of MK,which was recently found in various human tumorcell lines, was detected in 6 of 26cancerous tissues but not in non-cancerous tissues. [ABSTRACT FROM AUTHOR]

Human retinoblastoma (Rb) protein, immunopurified from an extract of recombinant baculovirus infected cells, stimulated 10 – 100-fold the activity of DNA polymerase α from calf thymus or human HeLa cells. Purified Rb protein is composed of two electrophoretically distinguishable forms, i.e., partially phosphorylated and under-phosphorylated forms. Dephosphorylation of Rb protein by protein phosphatase 2A largely diminished its stimulatory effect. On the other hand, a hyperphosphorylated Rb protein, obtained from insect cells overexpressing Rb protein, cyclin E and cyclin-dependent kinase 2 simultaneously, stimulated DNA polymerase α more strongly than the singly-expressed Rb protein. These results indicate that the phosphorylation is crucial for the stimulation. Rb protein isolated from human Burkitt lymphoma Raji cells also stimulated DNA polymerase α. In contrast, Rb protein did not affect eukaryotic DNA primase or Klenow fragment of Escherichia coli DNA polymerase I. By immunoprecipitation using anti-DNA polymerase α antibody, Rb protein in nuclear extract of Raji cells was co-precipitated with DNA polymerase α. This result indicates that DNA polymerase α exists as a complex containing phosphorylated Rb protein in cells. DNA polymerase α specifically bound to a purified Rb protein-immobilized Sepharose column. Rb protein also bound to DNA polymerase α trapped to anti-DNA polymerase α antibody-Sepharose column, suggesting the direct association of these two proteins. These observations suggest a new function of phosphorylated Rb protein in the regulation of DNA replication. [ABSTRACT FROM AUTHOR]

BCL-6: gene alterations have been observed in 27 – 45% of diffuse large B-cell lymphomas (DLBs) with chromosomal translocations at 3q27. The deregulated expression of normal BCL-6 protein caused by this chromosomal translocation is believed to be responsible for lymphomagenesis. Recently, we demonstrated that BCL-6 is expressed at high levels in germinal center B-cells as a 92-98 kDa nuclear protein in a constitutively phosphorylated form. In this study, we show that BCL-6 is phosphorylated by mitogen-activated protein kinase (MAPK) in vitro at the sites phosphorylated in vivo. These numerous phosphorylation sites were found to be located in its serine- and proline-clustered (SPC) region (amino acids-250-483). BCL-6 phosphorylation significantly increased in Ramos cells following stimulation with 12-o-tetradecanoylphorbol-13-acetate (TPA) or BCL-6- and erk1-transfected COS-7 cells stimulated with epidermal growth factor (EGF), and the increase of phosphorylation was inhibited by MEK1 inhibitor, PD98059. Furthermore, we observed that BCL-6 was associated with MAPK in vivo and its SPC region was important for this association. These results suggest that the functions of BCL-6 are regulated by phosphorylation mediated by the MAPK signaling pathway. [ABSTRACT FROM AUTHOR]

Abstract The human homologue of Drosophila tumor suppressor dlg, hDLG1, is one of the proteins known to interact with APC, a tumor suppressor for colorectal cancer. Alternative splicing of this gene generates transcripts either with [insertion 1 (I1)] or without 99 nucleotides in the 5' part of the dlg homology repeats (DHR) domain. We found almost equivalent expression of these two splicing variants in most human tissues; however, in skeletal muscle the transcript with the 99-bp insertion was predominant, and in the brain, that without the 99-bp insertion was expressed predominantly. We also examined alternative splicing in the region between the SH3 and GUK domains where two different sizes of insertions, 34 nucleotides (I2) or 100 nucleotides (I3), had been reported, and found various splicing patterns among the tissues examined. In brain we detected six different, alternatively spliced transcripts, two of which included a novel, 36-bp, brain-specific exon encoding a peptide bearing significant homology to a portion of rat synapse-associated protein, SAP97/PSD95. Subsequently, we investigated the splicing patterns of the hDLG1 gene in 24 neuroblastoma cell lines. In two-thirds of these lines, the splicing patterns were altered from those observed in normal brain tissue. As one-third retained the normal brain-splicing pattern, the loss of normal splicing of hDLG1 may not in itself cause formation of tumors, but it might reflect the biological character of individual neuroblastomas. [ABSTRACT FROM AUTHOR]

The MCC (mutated in colorectal cancer) gene product was localized to the terminal web and microvilli and along the lateral plasma membranes in colon epithelial cells. In the cerebellum, the MCC protein was highly expressed in the molecular layer, and was associated with the plasma membrane and membrane organelles in neuronal components. In COS-7 cells overexpressing exogenous MCC, this protein was not only associated with the plasma membrane and membrane organelles but was also distributed throughout their cytoplasm. After permeabilization of the COS-7 cells, the MCC protein that remained in the cell was still associated with the plasma membrane and membrane organelles. These results indicate that the MCC protein binds to both plasma membrane and membrane organelles. [ABSTRACT FROM AUTHOR]

The transcription factor GATA6 is a critical regulator of cell proliferation and development in the gastrointestinal tract. We have recently reported that GATA6 induces the expression of the intestinal stem cell marker LGR5 and enhances the clonogenicity and tumorigenicity of colon cancer cells, but not the growth of these cells cultured under adherent conditions. Here we show that REG4, a member of the regenerating islet-derived (REG) family, is also a target of GATA6. We further demonstrate that REG4 is downregulated by overexpression of miR-363, which suppresses GATA6 expression. Moreover, we show that GATA6-mediated activation of REG4 enhances the growth of colon cancer cells under adherent conditions and is required for their tumorigenicity. Taken together, our findings demonstrate that GATA6 simultaneously induces the expression of genes essential for the growth of colon cancer cells under adherent conditions (REG4) and genes required for their clonogenicity (LGR5), and that the miR-363-GATA6-REG4/LGR5 signaling cascade promotes the tumorigenicity of colon cancer cells. [ABSTRACT FROM AUTHOR]

The BCL-6 proto-oncogene encodes a 92- to 98-kDa transcriptional repressor containing the BTB/POZ domain at its N-terminal region and the zinc finger domain at its C-terminal region, respectively. In the present study, we examined the function of BCL-6 by using a recombinant adenovirus expressing BCL-6 (Ax1CA-BCL-6) and the lacZ reporter gene (Ax1CA-lacZ). Viability of CV-1 and HeLa cells infected with Ax1CA-BCL-6 was markedly reduced due to apoptosis, suggesting that BCL-6-overexpression induces apoptosis in CV-1 and HeLa cells. FACS analysis revealed that BCL-6-overexpressing cells are accumulated not only at the sub-G1 but also at G2/M phase. Induction of apoptosis by BCL-6 was preceded by down-regulation of apoptosis repressors BCL-2 and BCL-XL. These results suggest that BCL-6 induces apoptosis by regulating the expression of these apoptosis-regulating genes. [ABSTRACT FROM AUTHOR]