Predatory salamanders reverse the outcome of competition among three species of anuran tadpoles.

The outcome of competition among three species of anuran tadpoles in replicated artificial pond communities depends on the density of predatory salamanders present in the community. Predators differentially affect the survival of anuran species to metamorphosis and reverse the pattern of anuran relative abundance resulting from interspecific competition among tadpoles in the absence of predators.

Constitutive transcriptional activation by a beta-catenin-Tcf complex in APC-/- colon carcinoma.

The adenomatous polyposis coli (APC) tumor suppressor protein binds to beta-catenin, a protein recently shown to interact with Tcf and Lef transcription factors. The gene encoding hTcf-4, a Tcf family member that is expressed in colonic epithelium, was cloned and characterized. hTcf-4 transactivates transcription only when associated with beta-catenin. Nuclei of APC-/- colon carcinoma cells were found to contain a stable beta-catenin-hTcf-4 complex that was constitutively active, as measured by transcription of a Tcf reporter gene. Reintroduction of APC removed beta-catenin from hTcf-4 and abrogated the transcriptional transactivation. Constitutive transcription of Tcf target genes, caused by loss of APC function, may be a crucial event in the early transformation of colonic epithelium.

Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC.

Inactivation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates colorectal neoplasia. One of the biochemical activities associated with the APC protein is down-regulation of transcriptional activation mediated by beta-catenin and T cell transcription factor 4 (Tcf-4). The protein products of mutant APC genes present in colorectal tumors were found to be defective in this activity. Furthermore, colorectal tumors with intact APC genes were found to contain activating mutations of beta-catenin that altered functionally significant phosphorylation sites. These results indicate that regulation of beta-catenin is critical to APC's tumor suppressive effect and that this regulation can be circumvented by mutations in either APC or beta-catenin.

Identification of c-MYC as a target of the APC pathway.

The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. Here, the c-MYC oncogene is identified as a target gene in this signaling pathway. Expression of c-MYC was shown to be repressed by wild-type APC and activated by beta-catenin, and these effects were mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers.

Gene expression response to cisplatin treatment in drug-sensitive and drug-resistant ovarian cancer cells.

The molecular pathways activated in response to acute cisplatin exposure, as well as the mechanisms involved in the long-term development of cisplatin-resistant cancer cells remain unclear. Using whole genome oligonucleotide microarrays, we have examined the kinetics of gene expression changes in a cisplatin-sensitive cell line, A2780, and its cisplatin-resistant derivative, ACRP. Both sensitive and resistant cell lines exhibited a very similar response of p53-inducible genes as early as 16 h after treatment. This p53 response was further increased at the 24-h time point. These experiments identify p53 as the main pathway producing a large-scale transcriptional response after cisplatin treatment in these cells containing wild-type p53. Consistent with a role for the p53 response in cisplatin sensitivity, knockdown of the p53 protein with small interfering RNA led to a twofold decrease in cell survival in the resistant cells. In addition, our analysis also allowed the identification of several genes that are differentially expressed between sensitive and resistant cells. These genes include GJA1 (encoding connexin 43 (Cx43)) and TWIST1, which are highly upregulated in cisplatin-resistant cells. The importance of Cx43 in drug resistance was demonstrated through functional analyses, although paradoxically, inhibition of Cx43 function in high expressing cells led to an increase in drug resistance. The pathways important in cisplatin response, as well as the genes found differentially expressed between cisplatin-resistant and -sensitive cells, may represent targets for therapy aimed at reversing drug resistance.

Claudin-1 overexpression in melanoma is regulated by PKC and contributes to melanoma cell motility.

Serial analysis of gene expression followed by pathway analysis implicated the tight junction protein claudin-1 (CLDN1) in melanoma progression. Tight junction proteins regulate the paracellular transport of molecules, but staining of a tissue microarray revealed that claudin-1 was overexpressed in melanoma, and aberrantly expressed in the cytoplasm of malignant cells, suggesting a role other than transport. Indeed, melanoma cells in culture demonstrate no tight junction function. It has been shown that protein kinase C (PKC) can affect expression of claudin-1 in rat choroid plexus cells, and we observed a correlation between levels of activated PKC and claudin expression in our melanoma cells. To determine if PKC could affect the expression of CLDN1 in human melanoma, cells lacking endogenous claudin-1 were treated with 200 nM phorbol myristic acid (PMA). PKC activation by PMA caused an increase in CLDN1 transcription in 30 min, and an increase in claudin-1 protein by 12 h. Inhibition of PKC signaling in cells with high claudin-1 expression resulted in decreased claudin-1 expression. CLDN1 appears to contribute to melanoma cell invasion, as transient transfection of melanoma cells with CLDN1 increased metalloproteinase 2 (MMP-2) secretion and activation, and subsequently, motility of melanoma cells as demonstrated by wound-healing assays. Conversely, knockdown of CLDN1 by siRNA resulted in the inhibition of motility, as well as decreases in MMP-2 secretion and activation. These data implicate claudin-1 in melanoma progression.

The I kappa B proteins: members of a multifunctional family.

The I kappa B proteins bind to Rel/NF-kappa B transcription factors and modulate their activities. Although originally described only as cytoplasmic inhibitors of Rel/NF-kappa B transcription complexes, it is now clear that I kappa B proteins also have other functions.

Frequent silencing of the GPC3 gene in ovarian cancer cell lines.

GPC3 encodes a glypican integral membrane protein and is mutated in the Simpson-Golabi-Behmel syndrome. Simpson-Golabi-Behmel syndrome, an X-linked condition, is characterized by pre- and postnatal overgrowth as well as by various other abnormalities, including increased risk of embryonal tumors. The GPC3 gene is located at Xq26, a region frequently deleted in advanced ovarian cancers. To determine whether GPC3 is a tumor suppressor in ovarian neoplasia, we studied its expression and mutational status in 13 ovarian cancer cell lines. No mutations were found in GPC3, but its expression was lost in four (31%) of the cell lines analyzed. In an of the cases where GPC3 expression was lost, the GPC3 promoter was hypermethylated, as demonstrated by Southern analysis. Expression of GPC3 was restored by treatment of the cells with the demethylating agent 5-aza-2'-deoxycytidine. A colony-forming assay confirmed that ectopic GPC3 expression inhibited the growth of ovarian cancer cell lines. Our results show that GPC3, a gene involved in the control of organ growth, is frequently inactivated in a subset of ovarian cancers and suggest that it may function as a tumor suppressor in the ovary.

Truncation of the extracellular region abrogrates cell contact but retains the growth-suppressive activity of E-cadherin.

E-cadherin has been demonstrated to induce growth suppression and decrease the invasiveness of cancer cells and thus has been proposed to be a tumor suppressor gene. The ability of E-cadherin to mediate cell-cell contact and contact inhibition presumably accounts for its antitumor effects, which are attributed to the extracellular domain of the protein. Here we report that blocking the ability of E-cadherin to mediate contact inhibition by either antagonistic antibodies or expression of a mutant form of E-cadherin with the extracellular region deleted does not abrogate growth suppression. Transfection of the E-cadherin gene into the human prostate cancer cell line TSU.Pr-1 induced cell-cell contact formation, growth suppression, and redistribution of beta-catenin to the cell membrane. Treatment of the E-cadherin transfectant (CAD) with blocking antibodies disrupted cell-cell contact formation but did not influence the growth rate, suggesting that cell-cell interaction is not required for E-cadherin-mediated growth suppression. Similarly, transfection of an E-cadherin construct in which the NH2-terminal (extracellular) region was deleted did not allow cell-cell contact formation but induced growth suppression. In contrast, transfection of an E-cadherin construct in which the COOH-terminal (cytoplasmic) region was deleted did not induce suppression but promoted cell contact formation. In cells expressing E-cadherin lacking the cytoplasmic region, beta-catenin was evenly distributed in the cytoplasm. By contrast, in cells expressing E-cadherin lacking the extracellular region, beta-catenin was cell membrane associated. Growth suppression was always associated with the localization of beta-catenin to the cell membrane. The redistribution of beta-catenin from the cytoplasm to the cell membrane initially suggested the involvement of the Wnt signaling pathway in regulating cell growth. However, only small differences in beta-catenin/T-cell factor signaling were detected in control and E-cadherin-expressing cells, suggesting that the Wnt pathway is not involved. Taken together, these findings suggest that E-cadherin-induced growth inhibition may not be solely attributed to contact inhibition but may involve the redistribution of beta-catenin from the cytoplasm to the cell membrane, and this redistribution may affect growth pathways independent of T-cell factor.

Mutational analysis of the APC/beta-catenin/Tcf pathway in colorectal cancer.

Mutation of the adenomatous polyposis coli (APC) tumor suppressor gene initiates the majority of colorectal (CR) cancers. One consequence of this inactivation is constitutive activation of beta-catenin/Tcf-mediated transcription. To further explore the role of the APC/beta-catenin/Tcf pathway in CR tumorigenesis, we searched for mutations in genes implicated in this pathway in CR tumors lacking APC mutations. No mutations of the gamma-catenin (CTNNG1), GSK-3alpha (GSK3A), or GSK-3beta (GSK3B) genes were detected. In contrast, mutations in the NH2-terminal regulatory domain of beta-catenin (CTNNB1) were found in 13 of 27 (48%) CR tumors lacking APC mutations. Mutations in the beta-catenin regulatory domain and APC were observed to be mutually exclusive, consistent with their equivalent effects on beta-catenin stability and Tcf transactivation. In addition, we found that CTNNB1 mutations can occur in the early, adenomatous stage of CR neoplasia, as has been observed previously with APC mutations. These results suggest that CTNNB1 mutations can uniquely substitute for APC mutations in CR tumors and that beta-catenin signaling plays a critical role in CR tumorigenesis.

Coordinately up-regulated genes in ovarian cancer.

A better understanding of the molecular circuitry in normal ovarian tissues and in ovarian cancer will likely provide new targets for diagnosis and therapy. Recently, much has been learned about the genes expressed in ovarian cancer through studies with cDNA arrays and serial analysis of gene expression. However, these methods do not allow highly quantitative analysis of gene expression on a large number of specimens. Here, we have used quantitative real-time RT-PCR in a panel of 39 microdissected ovarian carcinomas of various subtypes to systematically analyze the expression of 13 genes, many of which were previously identified as up-regulated in a subset of ovarian cancers by serial analyses of gene expression. The genes analyzed are glutathione peroxidase 3 (GPX3), apolipoprotein J/clusterin, insulin-like growth factor-binding protein 2, epithelial cell adhesion molecule/GA733-2, Kop protease inhibitor, matrix gla protein, tissue inhibitor of metalloproteinase 3, folate receptor 1, S100A2, signal transducer and activator of transcription 1, secretory leukocyte protease inhibitor, apolipoprotein E, and ceruloplasmin. All of the genes were found overexpressed, some at extremely high levels, in the vast majority of ovarian carcinomas irrespective of the subtype. Interestingly, GPX3 was found at much higher levels in tumors with clear cell histology and may represent a biomarker for this subtype. Some of the genes studied here may thus represent targets for early detection ovarian cancer. The gene expression patterns were not associated with age at diagnosis, stage, or K-ras mutation status in ovarian cancer. We find that several genes are coordinately regulated in ovarian cancer, likely representing the fact that many genes are activated as part of common signaling pathways or that extensive cross-talk exists between several pathways in ovarian cancer. A statistical analysis shows that genes commonly up-regulated in ovarian cancer may result from the aberrant activation of a limited number of pathways, providing promising targets for novel therapeutic strategies.

Large-scale serial analysis of gene expression reveals genes differentially expressed in ovarian cancer.

Difficulties in the detection, diagnosis, and treatment of ovarian cancer result in an overall low survival rate of women with this disease. A better understanding of the pathways involved in ovarian tumorigenesis will likely provide new targets for early and effective intervention. Here, we have used serial analysis of gene expression (SAGE) to generate global gene expression profiles from various ovarian cell lines and tissues, including primary cancers, ovarian surface epithelia cells, and cystadenoma cells. The profiles were used to compare overall patterns of gene expression and to identify differentially expressed genes. We have sequenced a total of 385,000 tags, yielding >56,000 genes expressed in 10 different libraries derived from ovarian tissues. In general, ovarian cancer cell lines showed relatively high levels of similarity to libraries from other cancer cell lines, regardless of the tissue of origin (ovarian or colon), indicating that these lines had lost many of their tissue-specific expression patterns. In contrast, immortalized ovarian surface epithelia and ovarian cystadenoma cells showed much higher similarity to primary ovarian carcinomas than to primary colon carcinomas. Primary tissue specimens therefore appeared to be a better model for gene expression analyses. Using the expression profiles described above and stringent selection criteria, we have identified a number of genes highly differentially expressed between nontransformed ovarian epithelia and ovarian carcinomas. Some of the genes identified are already known to be overexpressed in ovarian cancer, but several represent novel candidates. Many of the genes up-regulated in ovarian cancer represent surface or secreted proteins such as claudin-3 and -4, HE4, mucin-1, epithelial cellular adhesion molecule, and mesothelin. Interestingly, both apolipoprotein E (ApoE) and ApoJ, two proteins involved in lipid homeostasis, are among the genes highly up-regulated in ovarian cancer. Selected serial analysis of gene expression results were further validated through immunohistochemical analysis of ApoJ, claudin-3, claudin-4, and epithelial cellular adhesion molecule in archival material. These experiments provided additional evidence of the relevance of our findings in vivo. The publicly available expression data reported here should stimulate and aid further research in the field of ovarian cancer.

A public database for gene expression in human cancers.

A public database, SAGEmap, was created as a component of the Cancer Genome Anatomy Project to provide a central location for depositing, retrieving, and analyzing human gene expression data. This database uses serial analysis of gene expression to quantify transcript levels in both malignant and normal human tissues. By accessing SAGEmap (http://www.ncbi.nlm.nih.gov/SAGE) the user can compare transcript populations between any of the posted libraries. As an initial demonstration of the database's utility, gene expression in human glioblastomas was compared with that of normal brain white matter. Of the 47,174 unique transcripts expressed in these two tissues, 471 (1.0%) were differentially expressed by more than 5-fold (P<0.001). Classification of these genes revealed functions consistent with the biological properties of glioblastomas, in particular: angiogenesis, transcription, and cell cycle related genes.

CDX2 is mutated in a colorectal cancer with normal APC/beta-catenin signaling.

The majority of human colorectal cancers have elevated beta-catenin/TCF regulated transcription due to either inactivating mutations of the APC tumor suppressor gene or activating mutations of beta-catenin. Surprisingly, one commonly used colorectal cancer cell line was found to have intact APC and beta-catenin and no demonstrable beta-catenin/TCF regulated transcription. However, this line did possess a truncating mutation in one allele of CDX2, a gene whose inactivation has recently been shown to cause colon tumorigenesis in mice. Expression of CDX2 was found to be induced by restoring expression of wild type APC in a colorectal cancer cell line. These findings raise the intriguing possibility that CDX2 contributes to APC's tumor suppressive effects.

The Yin-Yang of TCF/beta-catenin signaling.

Wingless/Wnt signaling directs cell-fate choices during embryonic development. In Drosophila, Wingless signaling mediates endoderm induction and the establishment of segment polarity in the developing embryo. The fly Wingless cascade is strikingly similar to the vertebrate Wnt signaling pathway, which controls a number of key developmental decisions such as dorsal-ventral patterning in Xenopus. Factors of the TCF/LEF HMG domain family (Tcfs) have recently been established as the downstream effectors of the Wingless/Wnt signal transduction pathways. Upon Wingless/Wnt signaling, a cascade is initiated that results in the accumulation of cytoplasmic beta-catenin (or its fly homolog, Armadillo). There is also a concomitant translocation of beta-catenin/Armadillo to the nucleus, where it interacts with a specific sequence motif at the N terminus of Tcfs to generate a transcriptionally active complex. This bipartite transcription factor is targeted to the upstream regulatory regions of Tcf target genes including Siamois and Nodal related gene-3 in Xenopus, engrailed and Ultrabithorax in Drosophila via the sequence-specific HMG box, and mediates their transcriptional activation by virtue of transactivation domains contributed by beta-catenin/Armadillo. In the absence of Wingless/Wnt signals, a key negative regulator of the pathway, GSK3 beta, is activated, which mediates the downregulation of cytoplasmic beta-catenin/Armadillo via the ubiquitin-proteasome pathway. In the absence of nuclear beta-catenin, the Tcfs recruit the corepressor protein Groucho to the target gene enhancers and actively repress their transcription. An additional corepressor protein, CREB-binding protein (CBP), may also be involved in this repression of Tcf target gene activity. Several other proteins, including adenomatous polyposis coli (APC), GSK3 beta, and Axin/Conductin, are instrumental in the regulation of beta-catenin/Armadillo. In APC-deficient colon carcinoma cell lines, beta-catenin accumulates and is constitutively complexed with nuclear Tcf-4. A proportion of APC wild-type colon carcinomas and melanomas also contains constitutive nuclear Tcf-4/beta-catenin complexes as a result of dominant mutations in the N terminus of beta-catenin that render it insensitive to downregulation by APC, GSK3 beta, and Axin/Conductin. This results in the unregulated expression of Tcf-4 target genes such as c-myc. Based on the established role for Tcf-4 in maintaining intestinal stem cells it is likely that deregulation of c-myc expression as a result of constitutive Tcf-4/beta-catenin activity promotes uncontrolled intestinal cell proliferation. This would readily explain the formation of intestinal polyps during colon carcinogenesis. Similar mechanisms leading to deregulation of Tcf target gene activity are likely to be involved in melanoma and other forms of cancer.

Kinesin is rapidly transported in the optic nerve as a membrane associated protein.

We have investigated the membrane vs. cytosolic distribution of newly synthesized and total kinesin in rabbit retinal ganglion cell axons which comprise the optic nerve. We find that kinesin is rapidly transported into the axon and that this newly synthesized protein is completely membrane-associated while approximately two third of the total kinesin in the optic nerve is membrane associated. Of this membrane associated kinesin about half is resistant to removal by treatment with 100 mM Na2CO3 (pH 11.3) and none can be stripped by 1 M NaCl. The newly synthesized axonal kinesin is completely resistant to removal by Na2CO3 treatment. By these criteria, at least one third of the total and essentially all of the rapidly transported axonal kinesin appears to exist as an integral membrane protein, consistent with it functioning as the anterograde motor for rapid vesicle transport from the cell body through the axon.

AAT1, a gene encoding a mitochondrial aspartate aminotransferase in Saccharomyces cerevisiae.

We have isolated a gene, AAT1, encoding an aspartate aminotransferase (AspAT) from a Saccharomyces cerevisiae genomic library. AAT1 encodes a 451 amino acid protein with a predicted molecular weight of 51,687, which is likely to be the yeast mitochondrial AspAT. Sequence comparison of this yeast AspAT with AspATs from other organisms shows a high degree of homology in regions previously shown to be important for catalysis. However, the yeast mitochondrial AspAT contains four obvious insertions with respect to all other known AspATs, suggesting that the AAT1-encoded protein represents a distinct AspAT.

A novel homozygous deletion at chromosomal band 6q27 in an ovarian cancer cell line delineates the position of a putative tumor suppressor gene.

Chromosomal band 6q27 is believed to contain a tumor suppressor gene important in the development of several cancer types, including ovarian cancer. However, repeated efforts to identify a tumor suppressor gene in this region have been unsuccessful. Because homozygous deletions have been useful in the positional cloning of a number of tumor suppressor genes, we initiated a systematic search for such deletions in ovarian cancer cell lines using 6q microsatellite markers. One of the cell lines, OV167, was found to contain an 80 kb homozygous deletion encompassing marker D6S193 at 6q27 but excluding nearby marker D6S297. No known genes were present in the deleted region. Because the homozygous deletion might affect the expression of nearby genes, we analyzed the expression of the two closest known genes flanking the deletion, RNASE6PL and RSK-3. The expression of these genes were unaffected by the homozygous deletion, suggesting that the functional target of the deletion is located between these two genes. A search of the region against expressed sequence tag (EST) databases revealed that it contained four sets of expressed sequences. The first expressed sequences were derived from a LINE repetitive element and were considered unlikely to represent a tumor suppressor gene. The other expressed sequence tags identified did not show homology to known genes and are currently being investigated. This data may significantly reduce the magnitude of the search for the 6q tumor suppressor gene as it suggests a small area as a prime target for investigation.

Implantable artificial lung. Preliminary report.

The ultimate treatment of chronic respiratory insufficiency is pulmonary replacement by an artificial organ, homologous lung transplantation, or chronic paracorporeal respiratory supplementation. The woven capillary membrane oxygenator appears to be a major development toward implantable artificial organs. The four units tested are made up of screens 3.5 by 4.0 cm. of capillary tubing 0.3 mm. I.D. by 0.64 mm. O.D. assembled into rectangular blocks. Units made up by five, ten, twenty, and forty screens have been assembled and tested according to the protocol suggested by Galletti. The maximum oxygen transfer rate with blood was 48 ml. per minute per square meter. Water carbon dioxide transfer rate was 23.1 ml. per minute per square meter. The pressure drops in the liquid phase were 8.5, 15.3, 13.8, 17.6 mm. Hg at 1 L. per minute flow. These results indicate that the woven capillary membrane lung is an acceptably efficient oxygenator. The characteristics of design and performance suggest that this oxygenator can be made to be implanted into the chest or used as a paracorporeal respiratory assistance device.

Nature of deposits in a tubular membrane oxygenator after prolonged extracorporeal circulation: a scanning electron microscope study.

Although silicone fibers are among the most compatible with tissue and blood, numerous deposits are observed after their prolonged usage in a capillary membrane oxygenator, even when the blood has been properly heparinized. The scanning electron microscope (SEM) study shows that the morphology of these deposits varies greatly, depending upon the part of the unit from which the sample is taken. The area close to the inlet is the most severely affected. The outlet zone is affected to a lesser degree, and the areas in between are only slightly affected.