Frequent somatic mutation of the MTS1/CDK4I (multiple tumor suppressor/cyclin-dependent kinase 4 inhibitor) gene in esophageal squamous cell carcinoma

We previously reported frequent loss of heterozygosity on chromosome 9p in esophageal carcinomas and suggested that a tumor suppressor gene located on this chromosomal arm might be involved in development of these cancers. Since recently published studies have shown that a gene mapped on chromosome 9p21, MTS1/CDK4I (multiple tumor suppressor 1/cyclin-dependent kinase 4 inhibitor), is frequently mutated in various types of tumors, we chose to examine esophageal squamous cell carcinomas for mutations in this candidate gene. DNA sequence analyses revealed somatic mutations of MTS1/CDK4I in 14 of 27 tumors examined; 8 were frame-shift mutations and 6 were missense mutations. These results suggested that the MTS1/CDK4I gene is a tumor suppressor the inactivation of which plays an important role during carcinogenesis of the squamous cell type of esophageal carcinoma.     

Adenomatous polyposis coli protein is expressed in alternate stages of the ameloblast life cycle

Mutations of the adenomatous polyposis coli gene protein (APC) are associated with familial polyposis and also sporadic colon adenomas, both preconditions to cancer formation. Some familial polyposis patients also develop Gardner's syndrome, a condition characterized by supernumerary teeth, mandibular osteomas, and other maladies. We investigated participation of APC in normal tooth development. Using a monoclonal antibody to study APC expression in the forming rat incisor, we found no APC staining in differentiating ameloblasts, then strong staining in secreting ameloblasts and stratum intermedium cells, followed by cells in the transition stage which did not stain. Intense APC staining resumes in maturation-stage ameloblasts and proximal papillary cells. APC staining disappears again in reduced ameloblasts at the conclusion of amelogenesis. APC staining was not seen in any other odontogenic cells. We report a unique system in which APC expression is upregulated and downregulated twice during the normal life cycle of ameloblasts. APC, therefore, is important in the normal maturation of both colonic epithelium and odontogenic epithelium. At this point, we cannot rule out any of the known functions of APC, which include: modulation of cell adhesion by binding to catenin, regulation of beta-catenin as a differentiative signaling molecule, and promotion of microtubule assembly. In this respect, the rat incisor enamel organ provides a unique tissue for studying the regulation and functions of APC.     

Cellular expression of a GPI-linked T cell activation protein

A T cell activation protein was identified by generating a monoclonal antibody (anti-D2) against a gamma delta T cell receptor bearing gibbon ape T cell line (MLA144). Immunoprecipitation studies revealed three polypeptides of 180, 150, and 120 kDa. The antigen was also found to be expressed on endothelial cells in vivo and in vitro and on tumor cell lines from a variety of tissues. Studies performed using a variety of antibodies reveal this protein to be identical to an endothelial cell protein previously identified by several antibodies to T cell activation proteins (CDw109). We demonstrate that this protein is anchored in the membrane via a glycosylphosphatidylinositol (GPI) tail in T cells, tumor cells, and endothelial cells. An analysis of tissue sections reveals this protein to be normally highly expressed on vascular endothelial cells.     

Clonal deletion of V beta 5+ T cells by transgenic I-E restricted to thymic medullary epithelium

A variety of cell types expressing MHC class II molecules is known to function as APC in vitro. We employed the Ig kappa gene enhancer and promoter to target the class II E alpha gene, and thereby I-E, exclusively to B cells to address their APC function in vivo. Although transgenic I-E was expressed on B lymphocytes, we unexpectedly obtained I-E on thymic medullary epithelium but not macrophages and at low frequency on dendritic cells. Using these transgenic mice, we constructed bone marrow irradiation chimeras with I-E expressed only on medullary epithelium, in order to determine the role of this cell type in tolerance by clonal deletion in the thymus. Although it is accepted that bm-derived cells play a primary role in deletion, and thymic epithelium can delete clones to a lesser degree, the role of cortical vs medullary thymic epithelium has not been directly dissected. We demonstrate that medullary epithelium alone can tolerize by partial deletion of I-E-reactive V beta 5+ T cells. These results indicate a role for medullary epithelium in deletion during the later stages of thymic development, and support the notion that positive and negative selection of developing T cells can occur in distinct temporal and anatomic compartments.     

Localization and characterization of a chromosome 11 tumor suppressor gene using organotypic raft cultures

The development and progression of human cancer often involves the inactivation of tumor suppressor gene function. Alterations in human chromosome 11 during the development of human cutaneous squamous cell carcinoma suggest the presence of a tumor suppressor gene on this chromosome. Moreover, previous studies in our laboratory demonstrated the presence of a functional tumor suppressor gene on chromosome 11 for the human cutaneous squamous cell carcinoma cell line A388.6TG.c2. In this investigation, we have used organotypic culturing of epithelial cells as a novel in vitro assay for tumor suppression. A388.6TG.c2 and control cells form an abnormal stratified epithelium of 8-12 layers when cultivated on organotypic rafts. In contrast, the chromosome 11 microcell hybrids, HMC 100p4B and HMC 100p5A, form an epithelium of only two to three cell layers. This in vitro growth suppression of the chromosome 11 microcell hybrids in the organotypic rafts correlates well with our previous in vivo skin graft experiments. Comparison of the proliferation and apoptotic indices of cell lines grown on the organotypic rafts suggests that the tumor suppressor gene on chromosome 11 has restricted the ability of the microcell hybrids to stratify but has not significantly altered their ability to undergo cell division or programmed cell death. Furthermore, flow cytometric analysis of cells grown on organotypic raft cultures suggests that the chromosome 11 microcell hybrids are actively progressing through the cell cycle rather than arrested in a particular stage. We have used this novel application of organotypic raft cultures to further localize the chromosome 11 tumor suppressor gene. Introduction of a single der(11)t(X;11) chromosome lacking most of the long arm of chromosome 11 into A388.6TG.c2 does not affect growth on organotypic raft cultures. These data suggest the tumor suppressor gene maps to the long arm of chromosome 11 in the region of 11q13-qter.     

The fourth armadillo repeat of plakoglobin (gamma-catenin) is required for its high affinity binding to the cytoplasmic domains of E-cadherin and desmosomal cadherin Dsg2, and the tumor suppressor APC protein

Plakoglobin is a member of a protein family with a repeated amino acid motif, the armadillo repeat, and is a cytoplasmic protein found in both adherens junctions and desmosomes. Plakoglobin has been shown to form distinct complexes with cadherins or desmosomal cadherins. Also, plakoglobin has been shown to complex with APC, the tumor suppressor gene product. Recently we isolated a cDNA clone encoding plakoglobin lacking the fourth armadillo repeat of the original 13-repeat protein [Ozawa et al. (1995) J. Biochem. 118, 836-840]. In this study, we established an in vitro assay system to study the molecular interaction of plakoglobin with cadherins, the APC gene product, and alpha-catenin. Establishment of the system and cloning of an alternate form of plakoglobin cDNA allowed us to examine the biological activity of plakoglobin lacking the fourth armadillo repeat. Experiments with the bacterially expressed 12-repeat plakoglobin revealed that the protein binds to E-cadherin, desmoglein (Dsg2), and APC with lower affinity than the 13-repeat form does. Consistent with the observation that the affinity of alpha-catenin for these two alternate forms was similar, we found amino acid residues 104 to 145 of plakoglobin, the residues present in both isoforms, are sufficient for its binding to alpha-catenin.     

Calcium-activated potassium channel gene expression in the midgut of Drosophila

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.     

Identification of a domain of Axin that binds to the serine/threonine protein phosphatase 2A and a self-binding domain

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and beta-catenin. Axin has been shown to have distinct binding sites for GSK-3 and beta-catenin and to promote the phosphorylation of beta-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through beta-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of beta-catenin. Here, we report the results of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. We found that Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. Our results suggest that protein phosphatase 2A might interact with the Axin.APC.GSK-3.beta-catenin complex, where it could modulate the effect of GSK-3 on beta-catenin or other proteins in the complex. We also identified a region of Axin that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, we demonstrated that the C-terminal 100 amino acids of Axin could bind to the same region as other Axin molecules.     

Expression of p21WAF1 predicts outcome of esophageal cancer patients treated by surgery alone or by combined therapy modalities

The p21WAF1 protein is an important regulator of the cell cycle. Its expression and prognostic significance were investigated immunohistochemically in samples of normal esophageal squamous epithelium (n = 10), severe squamous cell dysplasia (n = 20), carcinoma in situ (n = 14), permanent esophageal squamous cell carcinoma cell lines (n = 3), and invasive squamous cell carcinomas treated either by potentially curative resection (n = 172) or by combined modality therapy (radiochemotherapy +/- surgery; n = 38). Whereas p21WAF1 expression in the normal epithelium was restricted to a few cells adjacent to the basal cell layer, p21WAF1 overexpression was frequently found in preneoplasias and invasive carcinomas. Expression of p21WAF1 in invasive carcinomas was not correlated with tumor differentiation, pT category, or pN category. Among carcinomas treated by potential curative resection, univariate (P = 0.0025) and multivariate (P = 0.0081) survival analysis showed significant correlation of strong p21WAF1 expression (> or =50% p21WAF1-positive tumor cells) with poor overall survival. Univariate survival analysis (P = 0.0006) revealed the same prognostic influence in the group of patients treated by combined modality therapy. We conclude that overexpression of p21WAF1 protein is a frequent event in preneoplasias and neoplasias of the esophagus. Immunohistochemical examination of p21WAF1 expression may provide important prognostic information for decision-making in the treatment of patients with esophageal cancer.     

Recombinant PML adenovirus suppresses growth and tumorigenicity of human breast cancer cells by inducing G1 cell cycle arrest and apoptosis

Our previous studies demonstrated that the promyelocytic leukemia gene, PML which involved in the 15;17 translocation in acute promyelocytic leukemia (APL) is a growth and transformation suppressor. In this study, recombinant PML adenovirus, Ad-PML was constructed and used to infect human breast cancer cells in vitro and in vivo, the anti-oncogenic function of PML and its mechanism of growth suppressing effect in breast cancer cells were examined. We showed that Ad-PML effectively infected the MCF-7 and SK-BR-3 cells. A high level of PML protein was expressed within 24 h post-infection and a detectable level remained at day 16. Ad-PML significantly suppressed the growth rate, clonogenicity, and tumorigenicity of breast cancer cells. Intratumoral injections of MCF-7-induced tumors by high titer Ad-PML suppressed tumor growth in nude mice by about 80%. The injection sites expressed high level of PML and associated with a massive apoptotic cell death. To elucidate the molecular mechanism of PML's growth suppressing function, we examined the effect of Ad-PML on cell cycle distribution in MCF-7 and SK-BR-3 cells. We found that Ad-PML infection caused a cell cycle arrest at the G1 phase. We further showed that G1 arrest of MCF-7 cells is associated with a significant decrease in cyclin D1 and CDK2. An increased expression of p53, p21 and cyclin E was found. The Rb protein became predominantly hypophosphorylated 48 h post-infection. These findings indicate that PML exerts its growth suppressing effects by modulating several key G1 regulatory proteins. Our study provides important insight into the mechanism of tumor suppressing function of PML and suggests a potential application of Ad-PML in human cancer gene therapy.     

Putative control of angiogenesis in hemangioblastomas by the von Hippel-Lindau tumor suppressor gene

The hypoxia-inducible endothelial cell-specific mitogen vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) is expressed in low amounts in adult human brain, but is highly upregulated in the perinecrotic palisading cells of glioblastomas. We observed high VEGF expression in cerebellar hemangioblastomas, which are highly vascular, nonnecrotic and presumably nonhypoxic tumors, and hypothesized that a mechanism other than hypoxia leads to VEGF upregulation. Because hemangioblastomas develop in patients with von Hippel-Lindau disease, and mutations of the von Hippel-Lindau tumor suppressor (VHL) gene have also been reported in sporadic hemangioblastomas, we investigated VHL expression in normal cerebellum and in hemangioblastomas and tested the hypothesis that mutations in the VHL gene lead to upregulation of VEGE We observed constitutive expression of VHL mRNA, but downregulation of VEGF mRNA in the postnatal cerebellum. In the adult cerebellum, VHL is predominantly expressed in neuronal cells. In hemangioblastomas, VHL expression appears to be restricted to stromal cells, suggesting that the neoplastic component is the stromal cell. VHL-deficient renal cell carcinoma cells (786-0) produced significantly higher levels of VEGF mRNA and protein compared with 786-0/ wt10 cells, which were stably transfected with the wild-type VHL gene. Our observations suggest that VHL mutations affect stromal cells in hemangioblastomas and that VEGF is upregulated in stromal cells as a consequence of mutations in the VHL gene.     

Cloning of a highly conserved human protein serine-threonine phosphatase gene from the glioma candidate region on chromosome 19q13.3

Allelic loss studies have suggested that a glioma tumor suppressor gene resides in a 425-kb region of chromosome 19q, telomeric to D19S219 and centromeric to D19S112. Exon amplification of a cosmid contig spanning this region yielded four exons with high homology to a rat protein serine-threonine phosphatase from a cosmid approximately 100 kb telomeric to D19S219. Isolation of a near full-length cDNA from a human fetal brain cDNA library revealed a protein serine-threonine phosphatase with a tetratricopeptide motif, almost identical to human PPP5C (PP5) and highly homologous to rat PPT. Northern blotting demonstrated expression in most tissues, including brain. Primary and cultured gliomas were studied for genetic alterations in this gene using pulsed-field gel electrophoresis, routine Southern blots, and genomic DNA-and RNA-based single-strand conformation polymorphism analysis. Genomic alterations were were not detected in any of the gliomas, and all studied gliomas expressed the gene, suggesting that this phosphatase is not the putative chromosome 19q glioma tumor suppressor gene.