Mutations in AXIN2 cause colorectal cancer with defective mismatch repair by activating beta-catenin/TCF signalling.
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Axin Protein↗
Biomedical subjects
J M Cunningham
Publications and source records attributed to J M Cunningham.
Induction of human fetal globin gene expression by a novel erythroid factor, NF-E4.
The stage selector protein (SSP) is a heteromeric complex involved in preferential expression of the human gamma-globin genes in fetal-erythroid cells. We have previously identified the ubiquitous transcription factor CP2 as a component of this complex. Using the protein dimerization domain of CP2 in a yeast two-hybrid screen, we have cloned a novel gene, NF-E4, encoding the tissue-restricted component of the SSP. NF-E4 and CP2 coimmunoprecipitate from extract derived from a fetal-erythroid cell line, and antiserum to NF-E4 ablates binding of the SSP to the gamma promoter. NF-E4 is expressed in fetal liver, cord blood, and bone marrow and in the K562 and HEL cell lines, which constitutively express the fetal globin genes. Enforced expression of NF-E4 in K562 cells and primary erythroid progenitors induces endogenous fetal globin gene expression, suggesting a possible strategy for therapeutic intervention in the hemoglobinopathies.
Amino Acid Sequence↗
An integrated somatic cell hybrid, YAC, and BAC map of the Rmc1 region of mouse chromosome 1.
Rmc1, the cellular receptor for the polytropic class of murine retroviruses, determines the tissue tropism of the virus and therefore plays a critical role in the pathogenesis of polytropic virus-induced leukemia. Previously we reported the physical mapping of this gene to a 5-cM region of mouse chromosome 1 and the construction of a yeast artificial chromosome (YAC) contig across this region. In this report we describe the refinement of the Rmc1 candidate region to approximately 600 kb and the generation of an integrated somatic cell hybrid, YAC, and bacterial artificial chromosome contig spanning the region. A number of genes and loci were physically ordered along the chromosome, including a recently identified candidate for Rmc1.
Animals↗
Glucose transporter Glut3 is targeted to secretory vesicles in neurons and PC12 cells.
In rat brain and cultured neuroendocrine PC12 cells, Glut3 is localized at the cell surface and, also, in a distinct population of homogenous synaptic-like vesicles. Glut3-containing vesicles co-purify with "classical" synaptic vesicles, but can be separated from the latter by sucrose gradient centrifugation. Unlike classical synaptic vesicles, Glut3-containing vesicles possess a high level of aminopeptidase activity, which has been identified as aminopeptidase B. This enzyme has recently been shown to be a marker of the secretory pathway in PC12 cells (Balogh, A., Cadel, S., Foulon, T., Picart, R., Der Garabedian, A., Rousselet, A., Tougard, C., and Cohen, P. (1998) J. Cell Sci. 111, 161-169). We, therefore, conclude that Glut3 is targeted to secretory vesicles in both neurons and PC12 cells.
Animals↗
Genetic analysis of collagen Q: roles in acetylcholinesterase and butyrylcholinesterase assembly and in synaptic structure and function.
Acetylcholinesterase (AChE) occurs in both asymmetric forms, covalently associated with a collagenous subunit called Q (ColQ), and globular forms that may be either soluble or membrane associated. At the skeletal neuromuscular junction, asymmetric AChE is anchored to the basal lamina of the synaptic cleft, where it hydrolyzes acetylcholine to terminate synaptic transmission. AChE has also been hypothesized to play developmental roles in the nervous system, and ColQ is also expressed in some AChE-poor tissues. To seek roles of ColQ and AChE at synapses and elsewhere, we generated ColQ-deficient mutant mice. ColQ-/- mice completely lacked asymmetric AChE in skeletal and cardiac muscles and brain; they also lacked asymmetric forms of the AChE homologue, butyrylcholinesterase. Thus, products of the ColQ gene are required for assembly of all detectable asymmetric AChE and butyrylcholinesterase. Surprisingly, globular AChE tetramers were also absent from neonatal ColQ-/- muscles, suggesting a role for the ColQ gene in assembly or stabilization of AChE forms that do not themselves contain a collagenous subunit. Histochemical, immunohistochemical, toxicological, and electrophysiological assays all indicated absence of AChE at ColQ-/- neuromuscular junctions. Nonetheless, neuromuscular function was initially robust, demonstrating that AChE and ColQ do not play obligatory roles in early phases of synaptogenesis. Moreover, because acute inhibition of synaptic AChE is fatal to normal animals, there must be compensatory mechanisms in the mutant that allow the synapse to function in the chronic absence of AChE. One structural mechanism appears to be a partial ensheathment of nerve terminals by Schwann cells. Compensation was incomplete, however, as animals lacking ColQ and synaptic AChE failed to thrive and most died before they reached maturity.
Acetylcholinesterase↗
Enforced expression of the GATA-2 transcription factor blocks normal hematopoiesis.
The zinc finger transcription factor GATA-2 is highly expressed in immature hematopoietic cells and declines with blood cell maturation. To investigate its role in normal adult hematopoiesis, a bicistronic retroviral vector encoding GATA-2 and the green fluorescent protein (GFP) was used to maintain the high levels of GATA-2 that are normally present in primitive hematopoietic cells. Coexpression of the GFP marker facilitated identification and quantitation of vector-expressing cells. Bone marrow cells transduced with the GATA-2 vector expressed GFP as judged by flow cytometry and GATA-2 as assessed by immunoblot analysis. A 50% to 80% reduction in hematopoietic progenitor-derived colony formation was observed with GATA-2/GFP-transduced marrow, compared with marrow transduced with a GFP-containing vector lacking the GATA-2 cDNA. Culture of purified populations of GATA-2/GFP-expressing and nonexpressing cells confirmed a specific ablation of the colony-forming ability of GATA-2/GFP-expressing progenitor cells. Similarly, loss of spleen colony-forming ability was observed for GATA-2/GFP-expressing bone marrow cells. Despite enforced GATA-2 expression, marrow cells remained viable and were negative in assays to evaluate apoptosis. Although efficient transduction of primitive Sca-1(+) Lin- cells was observed with the GATA-2/GFP vector, GATA-2/GFP-expressing stem cells failed to substantially contribute to the multilineage hematopoietic reconstitution of transplanted mice. Additionally, mice transplanted with purified, GATA-2/GFP-expressing cells showed post-transplant cytopenias and decreased numbers of total and gene-modified bone marrow Sca-1(+) Lin- cells. Although Sca-1(+) Lin- bone marrow cells expressing the GATA-2/GFP vector were detected after transplantation, no appreciable expansion in their numbers occurred. In contrast, control GFP-expressing Sca-1(+) Lin- cells expanded at least 40-fold after transplantation. Thus, enforced expression of GATA-2 in pluripotent hematopoietic cells blocked both their amplification and differentiation. There appears to be a critical dose-dependent effect of GATA-2 on blood cell differentiation in that downregulation of GATA-2 expression is necessary for stem cells to contribute to hematopoiesis in vivo.
3T3 Cells↗
Insulitis and mechanisms of disease resistance: studies in an animal model of insulin dependent diabetes mellitus.
Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease characterised by extreme insulin deficiency due to an overall decrease in the mass of properly functioning beta-cells. This reduction occurs as a result of insulitis. the outcome of which will depend upon the intensity of the cytotoxic attack and the ability of beta-cells to resist and repair immune mediated cell damage. To further elucidate the relationship between the insulitis process and beta-cell defence and repair mechanisms in the prevention of diabetes we have studied a unique subgroup of diabetes prone (DP) BB/S rats which have demonstrated an ability to recover from IDDM (BB/S-R). Animals were diagnosed as diabetic at 115 days of age, subsequently receiving insulin therapy (1.49+/-0.1 IU/day) for a total of 19.7 days during 1 to 4 episodes of IDDM. Following a prolonged symptom-free period of 90 days, an IPGTT revealed that BB/S-R rats possessed normal glycaemic control. Islets were isolated from the BB/S-R rats and their glucose-stimulated insulin response was shown to be comparable to Wistar control islets. Furthermore, control and BB/S-R islets showed both a similar structural integrity and insulin content. BB/S-R islets cultured for 24 hr in IL-1beta (10(-13) M) maintained a significant insulin secretory response to glucose in contrast to Wistar controls in which the response was completely inhibited. Nitrite production was induced by IL-1beta, in a dose-dependent manner, in control islets whereas there was no significant increase in production in the islets of BB/S-R rats. These findings suggest that previous immune directed beta-cell attack may induce a state of increased resistance to subsequent deleterious effects of cytokine-mediated cytotoxicity. Overall therefore, the present study shows how the "recovered" BB/S-R rat model provides a unique opportunity to assess the direct effects of insulitis on pancreatic islets and how this interaction may subsequently determine disease outcome.
Age of Onset↗
Budd-Chiari syndrome caused by Gaucher's disease.
We present a unique case of Budd-Chiari syndrome caused by Gaucher's disease. The diagnosis was based on Doppler sonography, magnetic resonance imaging, contrast-enhanced three-dimensional magnetic resonance angiography, standard venography, and venous pressure measurements and was confirmed histologically.
Budd-Chiari Syndrome↗
Distribution of ten laminin chains in dystrophic and regenerating muscles.
Using immunohistochemical methods, we assessed the distribution of all 10 known laminin chains (alpha1-5, beta1-3, gamma1 and gamma2) in skeletal muscles from patients with Duchenne, congenital, limb girdle, or Emery-Dreifuss muscular dystrophies. The alpha2, beta1 and gamma1 chains were abundant in the basal lamina surrounding muscle fibers in normal controls; alpha1, alpha3-alpha5, beta3, and gamma2 were undetectable; and beta2 was present at a low level. Compared to controls, levels of the alpha5 chain were increased in muscles from many dystrophic patients; levels of beta1 were reduced and/or levels of beta2 were increased in a minority. However, these changes were neither specific for, nor consistent within, diagnostic categories. In contrast, levels of alpha4 were increased in muscles from all patients with alpha2 laminin (merosin)-deficient congenital muscular dystrophy. Loss of alpha2 laminin in congenital dystrophy is disease-specific but some other changes in laminin isoform expression in dystrophic muscles could be secondary consequences of myopathy, denervation, regeneration or immaturity. To distinguish among these possibilities, we compared the laminins of embryonic, denervated, regenerating, and mutant mouse muscles with those in normal adult muscle. Embryonic muscle basal lamina contained alpha4 and alpha5 along with alpha2, and regenerating muscle re-expressed alpha5 but not alpha4. Levels of alpha5 but not alpha4 were increased in dystrophin (mdx) mutants and in dystrophin/utrophin double mutants (mdx:utrn -/-), models for Duchenne dystrophy. In contrast, laminin alpha4 was upregulated more than alpha5 in muscles of laminin alpha2 mutant mice (dy/dy; a model for alpha2-deficient congenital dystrophy). Based on these results, we hypothesize that the expression of alpha5 in many dystrophies reflects the regenerative process, whereas the selective expression of alpha4 in alpha2-deficient muscle is a specific compensatory response to loss of alpha2.
Adolescent↗
Mapping of semantic, phonological, and orthographic verbal working memory in normal adults with functional magnetic resonance imaging.
Twelve neurologically normal participants (4 men and 8 women) performed semantic, phonological, and orthographic working memory tasks and a control task during functional magnetic resonance imaging. Divergent regions of the posterior left hemisphere used for decoding and storage of information emerged in each working memory versus control task comparison. These regions were consistent with previous literature on processing mechanisms for semantic, phonological, and orthographic information. Further, working memory versus control task differences extended into the left frontal lobe, including premotor cortex, and even into subcortical structures. Findings were consistent with R. C. Martin and C. Romani's (1994) contention that different forms of verbal working memory exist and further suggest that a reconceptualization of premotor cortex functions is needed.
Adolescent↗
Receptors for polytropic and xenotropic mouse leukaemia viruses encoded by a single gene at Rmc1.
The onset of leukaemia caused by type C retroviruses (MLV) in mice is accelerated by the emergence of recombinant polytropic or mink cell focus-forming (MCF) viruses. Susceptibility to infection by polytropic/MCF and also by closely related xenotropic MLV has been mapped to Rmc1 on mouse chromosome 1 (refs 5-7). To identify this gene, we introduced an expression cDNA library prepared from mouse NIH3T3 fibroblasts into nonpermissive hamster cells and screened these cells for acquired susceptibility to MCF viruses encoding beta-galactosidase and G418 resistance. From hamster cell clones identified in the screen, we recovered a mouse cDNA that maps to Rmc1 and confers MCF MLV infection when expressed in nonpermissive cell lines. It encodes a membrane protein related to Syg1p (suppressor of yeast G alpha deletion; ref. 8). The receptor-binding domain of the MCF MLV envelope protein binds specifically to Xenopus laevis oocytes that express mouse Syg1, suggesting it functions as a receptor that mediates virus entry. We also obtained the cDNA encoding human SYG1. When expressed in hamster cells, it establishes infectivity by MCF MLV as well as xenotropic MLV, which do not infect laboratory mice.
3T3 Cells↗
Identification of a receptor-binding pocket on the envelope protein of friend murine leukemia virus.
Based on previous structural and functional studies, a potential receptor-binding site composed of residues that form a pocket at one end of the two long antiparallel helices in the receptor-binding domain of Friend 57 murine leukemia virus envelope protein (RBD) has been proposed. To test this hypothesis, directed substitutions for residues in the pocket were introduced and consequences for infection and for receptor binding were measured. Receptor binding was measured initially by a sensitive assay based on coexpression of receptor and RBD in Xenopus oocytes, and the findings were confirmed by using purified proteins. Three residues that are critical for both binding and infection (S84, D86, and W102), with side chains that extend into the pocket, were identified. Moreover, when mCAT-1 was overexpressed, the infectivity of Fr57-MLV carrying pocket substitutions was partially restored. Substitutions for 18 adjacent residues and 11 other previously unexamined surface-exposed residues outside of the RBD pocket had no detectable effect on function. Taken together, these findings support a model in which the RBD pocket interacts directly with mCAT-1 (likely residues, Y235 and E237) and multiple receptor-envelope complexes are required to form the fusion pore.
3T3 Cells↗
Identification of Id2 as a globin regulatory protein by representational difference analysis of K562 cells induced to express gamma-globin with a fungal compound.
A fungus-derived compound (OSI-2040) which induces fetal globin expression in the absence of erythroid cell differentiation was identified in a high-throughput drug discovery program. We utilized this compound to isolate gamma-globin regulatory genes that are differentially expressed in OSI-2040-induced and uninduced cells in the human erythroleukemia cell line K562. Representational difference analysis (RDA) of cDNA revealed several genes that were significantly up- or down-regulated in OSI-2040-induced cells. One gene whose expression was markedly enhanced was the gene for the helix-loop-helix (HLH) transcription factor Id2. Southern analysis of RDA amplicons demonstrated progressive enrichment of Id2 with each successive subtraction of uninduced cDNA from induced cDNA. Northern analysis of OSI-2040-induced K562 cells confirmed that Id2 expression was directly up-regulated coordinately with gamma-globin. Analysis of other inducers of fetal globin demonstrated up-regulation of Id2 with sodium butyrate but not with hemin. Retrovirus-mediated overexpression of Id2 in K562 cells reproduced the enhancement of endogenous globin expression observed with OSI-2040 induction. Functional assays demonstrated that an E-box element in hypersensitivity site 2 is required for Id2-dependent enhancement of gamma-promoter activity. Protein binding studies suggest that alterations in E-box site occupancy by basic HLH proteins may influence this activity, thus expanding the potential role of these factors in globin gene regulation.
Blotting, Northern↗
Neural basis of endogenous and exogenous spatial orienting. A functional MRI study.
Whole-brain functional magnetic resonance imaging (MRI) was used to examine the neural substrates of internally (endogenous) and externally (exogenous) induced covert shifts of attention. Thirteen normal subjects performed three orienting conditions: endogenous (location of peripheral target predicted by a central arrow 80% of the time), exogenous (peripheral target preceded by noninformative central cue). Behavioral results indicated faster reaction times (RTs) for valid than for invalid trials for the endogenous condition but slower RTs for valid than for invalid trials for the exogenous condition (inhibition of return). The spatial extent and intensity of activation was greatest for the endogenous condition, consistent with the hypothesis that endogenous orienting is more effortful (less automatic) than exogenous orienting. Overall, we did not observe distinctly separable neural systems associated with the endogenous and exogenous orienting conditions. Both exogenous and endogenous orienting, but not the control condition, activated bilateral parietal and dorsal premotor regions, including the frontal eye fields. These results suggest a specific role for these regions in preparatory responding to peripheral stimuli. The right dorsolateral prefrontal cortex (BA 46) was activated selectively by the endogenous condition. This finding suggests that voluntary, but not reflexive, shifts of attention engage working memory systems.
Adult↗
Tumor suppressor gene alterations in malignant gliomas: histopathological associations and prognostic evaluation.
We have examined a series of 135 gliomas for alterations of the p53, CDKN2A (p16) and PTEN tumor suppressor genes (TSGs) in order to evaluate the incidence of their inactivation as a function of tumor malignancy and cellular differentiation, and to examine potential associations with patient outcome. The composition of this series, classified using WHO criteria, is as follows: 27 grade 2 tumors (11 astrocytomas, 12 oligoastrocytomas, 4 oligodendrogliomas), 42 grade 3 tumors (22 astrocytomas, 16 oligoastrocytomas, 4 oligodendrogliomas), and 66 grade 4 tumors (63 astrocytomas and 3 oligoastrocytomas). Similar frequencies of p53 mutation were observed among grade 2 (37.0%), and grade 3 tumors (38.1%), as well as between astrocytomas and mixed tumors. CDKN2A and PTEN mutations were clearly associated with increasing tumor malignancy (occurring in 0% of grade 2 tumors, 14.3% and 4.8% respectively of grade 3 tumors, and 27.3% and 30.3% respectively of grade 4 tumors) and were observed at substantially higher rates among astrocytomas. For the tumor suppressor genes examined, there was no relationship between the occurrence of any two TSG inactivation events. With regard to outcome, the p53 genetic status showed no significant relationship with patient survival. The CDKN2 and PTEN alterations were negative prognostic indicators of survival when evaluated in all 135 gliomas, but failed to predict outcome when evaluated in either of the high grade (3 or 4) tumor groups.
Adolescent↗
Reduced COX-2 protein in colorectal cancer with defective mismatch repair.
Most colorectal adenomas and carcinomas arise in the setting of chromosomal instability characterized by progressive loss of heterozygosity. In contrast, approximately 15-20% of colorectal neoplasms arise through a distinct genetic pathway characterized by microsatellite instability (MSI) associated with frequent loss of expression of one of the DNA mismatch repair enzymes, most often hMLH1 or hMSH2. These distinct genetic pathways are reflected by differences in tumor histopathology, distribution in the colon, prognosis, and dwell time required for progression from adenoma to carcinoma. To determine whether these two groups of tumors differ in their expression of cyclooxygenase-2 (COX-2), a putative chemopreventative target, immunostaining for this protein was performed in colorectal cancers categorized by the presence (n = 41) and absence (n = 66) of defective mismatch repair. Defective mismatch repair was defined by the presence of tumor microsatellite instability (MSI-H, > or =40% of markers demonstrating instability) and by the absence of protein expression for either hMLH1 or hMSH2. Overall, our results showed that low or absent COX-2 staining was significantly more common among tumors with defective mismatch repair (P = 0.001). Other features predictive of low COX-2 staining included marked tumor infiltrating lymphocytosis, and solid/cribiform or signet ring histological patterns. These observations indicate that colorectal cancers with molecular and phenotypic characteristics of defective DNA mismatch repair express lower levels of COX-2. The clinical implications of this biological distinction remain unknown but should be considered when assessing the efficacy of COX-2 inhibitors for chemoprevention in patients whose tumors may arise in the setting of defective DNA mismatch repair.
Adaptor Proteins, Signal Transducing↗
Hypermethylation of the hMLH1 promoter in colon cancer with microsatellite instability.
Recent studies have demonstrated the presence of microsatellite instability (MSI) in tumors from patients with hereditary nonpolyposis colon cancer and in a subset of patients with sporadic colorectal cancer (CRC). In sporadic CRC, three tumor phenotypes have been defined: microsatellite stable (MSS), low-frequency MSI, and high-frequency MSI (MSI-H). Although defective mismatch repair, consisting primarily of alterations in hMSH2 and hMLH1, is believed to be responsible for the MSI phenotype in the majority of patients with hereditary nonpolyposis colon cancer, the genetic defect responsible for this phenotype in sporadic CRC has yet to be clearly delineated. Somatic or germ-line alterations in these two genes have been identified in only a minority of these cases. Analysis of the protein expression patterns of hMSH2 and hMLH1 in unselected CRC, however, suggests that alterations in hMLH1 may account for a majority of the MSI-H cases. In an effort to explore the underlying molecular basis for these findings, we have examined the methylation status of the presumptive hMLHI promoter region in 31 tumors that vary in regard to their MSI status (MSI-H or MSS), their hMLH1 protein expression (MLH- or MLH+), and their gene mutation (Mut+ or Mut-) status. Hypermethylation of the hMLH1 promoter occurred in all 13 MSI-H/ MLH- tumors that did not have a detectable mutation within the hMLH1 gene. Of those MSI-H tumors containing germ-line or somatic alterations in hMLH1 (n = 7, including 3 frameshift, 1 nonsense, 2 missense mutations, and 1 tumor containing multiple mutations: missense, splice-site alteration, and a frameshift), four had a normal methylation pattern, whereas three others demonstrated hypermethylation of the hMLH1 promoter region. Two of these cases had a missense alteration, the other a frameshift alteration. The single MSI-H/Mut+ tumor that had normal hMLH1 and hMSH2 expression, as well as 9 of the 10 MSS cases, lacked methylation of the hMLH1 promoter. Hypermethylation of the hMSH2 promoter was not observed for any of the cases. These results suggest that hypermethylation of the hMLH1 promoter may be the principal mechanism of gene inactivation in sporadic CRC characterized by widespread MSI.
Adaptor Proteins, Signal Transducing↗
Search for chromosome instability in lymphocytes with germ-line mutations in DNA mismatch repair genes.
All colorectal cancer results from the accumulation of critical genetic alterations; however, the mechanisms of acquiring these mutations appear to be different in hereditary nonpolyposis colon cancer (HNPCC) and sporadic tumors with microsatellite instability compared with sporadic tumors with no microsatellite instability. To further explore the possible mechanisms of cancer predisposition in HNPCC, we studied chromosome breakage and induction of aneuploidy in the lymphoblastoid cell lines of four patients who were heterozygous for mutations in either hMSH2 or hMLH1--two components of the DNA mismatch repair complex. Cells were cultured under different stress systems, including exposure to bleomycin, mitomycin C, bromodeoxyuridine-induced sister chromatid exchange, and a DNA alkylator, N-methyl-N'-nitro-N-nitrosoguanidine. No significant differences were detected in the levels of major or minor chromosome breakage or aneuploidy compared with controls. These results suggest that a single "hit" to a DNA mismatch repair gene does not confer appreciable susceptibility to structural or numerical chromosomal alterations, which is consistent with previous observations that HNPCC-related cancers are more likely to be near diploid than are sporadic colon cancers.
Aneuploidy↗