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1.
Clin Genet ; 79(4): 321-8, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21204803

ABSTRACT

Lynch syndrome (LS) is the most common inherited form of colorectal cancer. Mutation carriers can reduce the morbidity and mortality associated with colorectal cancer through colonoscopy. Theoretical models suggest that such health-related behaviors might also bring psychological benefits. This study assessed whether colonoscopy following mutation detection was associated with the levels of depressive symptoms. Data were obtained from a prospective family cohort study offering genetic services for LS. Participants completed questionnaires prior to the provision of services and 6 months post-receipt of mutation results. One hundred thirty-four (134) persons were identified to carry a mutation and completed both the questionnaires. Main outcome measures were depressive symptoms 6 months post-receipt of test results. Mutation carriers who did not complete a colonoscopy within the 6 months following receipt of results were six times (p < 0.01; odds ratio = 6.06) more likely to report depressive symptoms at a level of clinical importance post-receipt of test results compared to those who did undergo colonoscopy. Facilitating the expeditious use of colonoscopy following mutation detection may benefit newly identified mutation carriers by addressing the objective risks for cancer and moderating underlying emotional distress responses to genetic risk information. Furthermore, depressive symptoms may interfere with behavioral compliance in some patients, suggesting referral to mental health specialists.


Subject(s)
Colonoscopy/methods , Colorectal Neoplasms, Hereditary Nonpolyposis/genetics , Genetic Testing/methods , Mutation , Adaptation, Psychological , Adaptor Proteins, Signal Transducing/genetics , Adolescent , Adult , Aged , Aged, 80 and over , Colorectal Neoplasms, Hereditary Nonpolyposis/diagnosis , Colorectal Neoplasms, Hereditary Nonpolyposis/psychology , DNA-Binding Proteins/genetics , Depression/psychology , Family Health , Female , Humans , Logistic Models , Male , Middle Aged , MutL Protein Homolog 1 , MutS Homolog 2 Protein/genetics , Nuclear Proteins/genetics , Outcome Assessment, Health Care , Prospective Studies , Surveys and Questionnaires , Young Adult
2.
Genesis ; 31(2): 72-7, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11668681

ABSTRACT

The Sil gene encodes a cytosolic protein required for mouse embryonic midline and left/right axial development. Based on the phenotype of Sil mutant embryos, we hypothesized that Sil may be required for the activity of Sonic Hedgehog (Shh), a secreted signaling molecule also critically important for the development of the embryonic axes and found mutated in multiple types of cancer. Here we tested the genetic interaction between Sil and the Shh pathway by generating and analyzing embryos carrying mutations in both Sil and Patched (Ptch), a Shh receptor that normally inhibits the signaling pathway in the absence of ligand and when mutated leads to constitutive activation of the pathway. We find that Sil(-/-) Ptch(-/-) embryos do not activate the Shh pathway and instead have a phenotype indistinguishable from Sil(-/-) embryos, in which there is a loss of activity of Shh. These results provide genetic evidence that Sil is an essential component of the Shh response, acting downstream to Ptch.


Subject(s)
Embryo, Mammalian/metabolism , Membrane Proteins/genetics , Oncogene Proteins, Fusion , Proteins/genetics , Proteins/metabolism , Signal Transduction , Trans-Activators/metabolism , Animals , Cell Death/genetics , Crosses, Genetic , Embryo, Mammalian/embryology , Epistasis, Genetic , Female , Gene Deletion , Gene Expression Regulation, Developmental , Genotype , Head/embryology , Hedgehog Proteins , In Situ Hybridization , In Situ Nick-End Labeling , Intracellular Signaling Peptides and Proteins , Male , Membrane Proteins/deficiency , Mice , Mice, Knockout , Patched Receptors , Patched-1 Receptor , RNA, Messenger/analysis , RNA, Messenger/genetics , Receptors, Cell Surface
3.
Environ Health Perspect ; 109(5): 495-500, 2001 May.
Article in English | MEDLINE | ID: mdl-11401761

ABSTRACT

Forest pesticide applicators constitute a unique pesticide use group. Aerial, mechanical-ground, and focal weed control by application of herbicides, in particular chlorophenoxy herbicides, yield diverse exposure scenarios. In the present work, we analyzed aberrations in G-banded chromosomes, reproductive hormone levels, and polymerase chain reaction-based V(D)J rearrangement frequencies in applicators whose exposures were mostly limited to chlorophenoxy herbicides. Data from appliers where chlorophenoxy use was less frequent were also examined. The biomarker outcome data were compared to urinary levels of 2,4-dichlorophenoxyacetic acid (2,4-D) obtained at the time of maximum 2,4-D use. Further comparisons of outcome data were made to the total volume of herbicides applied during the entire pesticide-use season.Twenty-four applicators and 15 minimally exposed foresters (control) subjects were studied. Categorized by applicator method, men who used a hand-held, backpack sprayer in their applications showed the highest average level (453.6 ppb) of 2,4-D in urine. Serum luteinizing hormone (LH) values were correlated with urinary 2,4-D levels, but follicle-stimulating hormone and free and total testosterone were not. At the height of the application season; 6/7 backpack sprayers, 3/4 applicators who used multinozzle mechanical (boom) sprayers, 4/8 aerial applicators, and 2/5 skidder-radiarc (closed cab) appliers had two or more V(D)J region rearrangements per microgram of DNA. Only 5 of 15 minimally exposed (control) foresters had two or more rearrangements, and 3 of these 5 subjects demonstrated detectable levels of 2,4-D in the urine. Only 8/24 DNA samples obtained from the exposed group 10 months or more after their last chlorophenoxy use had two rearrangements per microgram of DNA, suggesting that the exposure-related effects observed were reversible and temporary. Although urinary 2,4-D levels were not correlated with chromosome aberration frequency, chromosome aberration frequencies were correlated with the total volume of herbicides applied, including products other than 2,4-D. In summary, herbicide applicators with high urinary levels of 2,4-D (backpack and boom spray applications) exhibited elevated LH levels. They also exhibited altered genomic stability as measured by V(D)J rearrangement frequency, which appears reversible months after peak exposure. Though highly detailed, the limited sample size warrants cautious interpretation of the data.


Subject(s)
2,4-Dichlorophenoxyacetic Acid/urine , Forestry , Gonadal Steroid Hormones/urine , Herbicides/urine , Mutagenesis/drug effects , Pesticide Residues/adverse effects , 2,4-Dichlorophenoxyacetic Acid/adverse effects , Biomarkers/urine , Chromosome Aberrations , Dose-Response Relationship, Drug , Endocrine System/drug effects , Gene Rearrangement, T-Lymphocyte/drug effects , Gonadal Steroid Hormones/analysis , Herbicides/adverse effects , Humans , Male , Occupational Exposure/adverse effects , Occupational Exposure/analysis , Pesticide Residues/analysis , Receptors, Antigen, T-Cell/drug effects , T-Lymphocytes/drug effects
4.
J Biol Chem ; 276(31): 29126-33, 2001 Aug 03.
Article in English | MEDLINE | ID: mdl-11390401

ABSTRACT

Chromosomal translocations and deletions are among the major events that initiate neoplasia. For lymphoid chromosomal translocations, misrecognition by the RAG (recombination activating gene) complex of V(D)J recombination is one contributing factor that has long been proposed. The chromosomal translocations involving LMO2 (t(11;14)(p13;q11)), Ttg-1 (t(11;14)(p15;q11)), and Hox11 (t(10;14)(q24;q11)) are among the clearest examples in which it appears that a D or J segment has synapsed with an adventitious heptamer/nonamer at a gene outside of one of the antigen receptor loci. The interstitial deletion at 1p32 involving SIL (SCL-interrupting locus)/SCL (stem cell leukemia) is a case involving two non-V(D)J sites that have been suggested to be V(D)J recombination mistakes. Here we have used our human extrachromosomal substrate assay to formally test the hypothesis that these regions are V(D)J recombination misrecognition sites and, more importantly, to quantify their efficiency as V(D)J recombination targets within the cell. We find that the LMO2 fragile site functions as a 12-signal at an efficiency that is only 27-fold lower than that of a consensus 12-signal. The Ttg-1 site functions as a 23-signal at an efficiency 530-fold lower than that of a consensus 23-signal. Hox11 failed to undergo recombination as a 12- or 23-signal and was at least 20,000-fold less efficient than consensus signals. SIL has been predicted to function as a 12-signal and SCL as a 23-signal. However, we find that SIL actually functions as a 23-signal. These results provide a formal demonstration that certain chromosomal fragile sites can serve as RAG complex targets, and they determine whether these sites function as 12- versus 23-signals. These results quantify one of the three major factors that determine the frequency of these translocations in T-cell acute lymphocytic leukemia.


Subject(s)
Chromosomes, Human , DNA Nucleotidyltransferases/metabolism , DNA-Binding Proteins/genetics , Metalloproteins/genetics , Oncogene Proteins, Fusion , Transcription Factors , Translocation, Genetic , Adaptor Proteins, Signal Transducing , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , Chromosome Mapping , Chromosomes, Human, Pair 1 , Chromosomes, Human, Pair 10 , Chromosomes, Human, Pair 11 , Chromosomes, Human, Pair 14 , Consensus Sequence , Genes, RAG-1 , Homeodomain Proteins/genetics , Humans , Intracellular Signaling Peptides and Proteins , LIM Domain Proteins , Leukemia/genetics , Leukemia-Lymphoma, Adult T-Cell , Molecular Sequence Data , Oncogene Proteins/genetics , Polymerase Chain Reaction , Proteins/genetics , Proto-Oncogene Proteins/genetics , Recombination, Genetic , Sequence Deletion , T-Cell Acute Lymphocytic Leukemia Protein 1 , Tumor Cells, Cultured , VDJ Recombinases
5.
Nature ; 409(6822): 945-6, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11237017

ABSTRACT

Our sequence-tagged site-content map of chromosome 12 is now integrated with the whole-genome fingerprinting effort. It provides accurate and nearly complete bacterial clone coverage of chromosome 12. We propose that this integrated mapping protocol serves as a model for constructing physical maps for entire genomes.


Subject(s)
Chromosomes, Human, Pair 12 , Chromosomes, Artificial, Bacterial , Contig Mapping , Genome, Human , Humans , Sequence Tagged Sites
6.
Nature ; 409(6822): 953-8, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11237021

ABSTRACT

We have placed 7,600 cytogenetically defined landmarks on the draft sequence of the human genome to help with the characterization of genes altered by gross chromosomal aberrations that cause human disease. The landmarks are large-insert clones mapped to chromosome bands by fluorescence in situ hybridization. Each clone contains a sequence tag that is positioned on the genomic sequence. This genome-wide set of sequence-anchored clones allows structural and functional analyses of the genome. This resource represents the first comprehensive integration of cytogenetic, radiation hybrid, linkage and sequence maps of the human genome; provides an independent validation of the sequence map and framework for contig order and orientation; surveys the genome for large-scale duplications, which are likely to require special attention during sequence assembly; and allows a stringent assessment of sequence differences between the dark and light bands of chromosomes. It also provides insight into large-scale chromatin structure and the evolution of chromosomes and gene families and will accelerate our understanding of the molecular bases of human disease and cancer.


Subject(s)
Chromosome Aberrations , Genetic Markers , Genome, Human , Chromosome Mapping , Chromosomes, Artificial, Bacterial , Cytogenetic Analysis , Human Genome Project , Humans , In Situ Hybridization, Fluorescence , Radiation Hybrid Mapping , Sequence Tagged Sites
7.
Nucleic Acids Res ; 29(1): 144-7, 2001 Jan 01.
Article in English | MEDLINE | ID: mdl-11125073

ABSTRACT

GenMapDB (http://genomics.med.upenn.edu/genmapdb) is a repository of human bacterial artificial chromosome (BAC) clones mapped by our laboratory to sequence-tagged site markers. Currently, GenMapDB contains over 3000 mapped clones that span 19 chromosomes, chromosomes 2, 4, 5, 9-22, X and Y. This database provides positional information about human BAC clones from the RPCI-11 human male BAC library. It also contains restriction fragment analysis data and end sequences of the clones. GenMapDB is freely available to the public. The main purpose of GenMapDB is to organize the mapping data and to allow the research community to search for mapped BAC clones that can be used in gene mapping studies and chromosomal mutation analysis projects.


Subject(s)
Chromosome Mapping , DNA/genetics , Databases, Factual , Chromosomes, Artificial, Bacterial/genetics , Cloning, Molecular , Humans , Information Services , Internet , Sequence Tagged Sites
8.
Semin Hematol ; 37(4): 420-8, 2000 Oct.
Article in English | MEDLINE | ID: mdl-11071363

ABSTRACT

The National Cancer Institute has established an initiative, called the Cancer Chromosome Aberration Project (Ccap), in order to link and integrate the physical and genetic maps of the human genome with cytogenetic data and the location of chromosomal rearrangements in human diseases. This goal will be achieved by high-resolution fluorescence in situ hybridization (FISH) mapping of colony-purified bacterial artificial chromosome (BAC) clones spaced at 1-to 2-Mb intervals across the entire genome. All BAC clones will be anchored on the physical map by the presence of a mapped sequence tagged site (STS). The generation of a publicly accessible clone repository will allow convenient distribution of these BACs. Ccap data can be correlated with other cancer-associated and genomic databases, such as the catalog of chromosomal aberrations in cancer and the emerging full genomic sequence. We anticipate that the use of Ccap clones will expedite and refine the mapping of chromosomal breakpoints. The eventual set of approximately 3,000 Ccap BACs should facilitate the production of BAC-containing DNA chips for assessing copy number of genomic segments by matrix comparative genomic hybridization. In addition, the repository will provide genome-wide tools for defining chromosomal aberrations in cytological specimens by interphase cytogenetics. The Ccap Web site illustrates goals and progress of this initiative (http://www.ncbi.nlm.nih.gov/CCAP/).


Subject(s)
Chromosome Mapping , Databases, Factual/trends , Genome , Neoplasms/genetics , Cytogenetics , Forecasting , Humans
9.
Proc Natl Acad Sci U S A ; 97(7): 3497-502, 2000 Mar 28.
Article in English | MEDLINE | ID: mdl-10737801

ABSTRACT

We have cloned the genomic breakpoints for a balanced t(14;21)(q11. 2;q22) chromosomal translocation associated with T-cell acute lymphoblastic leukemia. Sequence analysis of the genomic breakpoints indicated that the translocation had been mediated by an illegitimate V(D)J recombination event that disrupted the T-cell receptor (TCR) alpha locus and placed the TCR alpha locus enhancer on the derivative 21 chromosome. We identified a previously unreported transcript, designated BHLHB1 (for basic domain, helix-loop-helix protein, class B, 1) that had been activated by the translocation. BHLHB1 mapped to the region of chromosome 21 that has been proposed to be responsible, at least in part, for the learning deficits seen in children with Down's syndrome. Although BHLHB1 expression normally is restricted to neural tissues, T-cell lymphoblasts with the t(14;21)(q11.2;q22) also expressed high levels of BHLHB1 mRNA. Expression of BHLHB1 dramatically inhibited E2A-mediated transcription activation in NIH 3T3 fibroblasts and Jurkat T cells. This observation suggests that BHLHB1, similar to SCL/TAL1, may exert a leukemogenic effect through a functional inactivation of E2A or related proteins.


Subject(s)
Chromosomes, Human , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Leukemia-Lymphoma, Adult T-Cell/genetics , Transcription Factors/genetics , Translocation, Genetic , Amino Acid Sequence , Base Sequence , Basic Helix-Loop-Helix Transcription Factors , Child , Female , Gene Rearrangement, alpha-Chain T-Cell Antigen Receptor , Humans , Molecular Sequence Data , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
11.
Genes Chromosomes Cancer ; 27(4): 418-23, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10719373

ABSTRACT

Genes that play roles in malignant transformation have often been found proximate to cancer-associated chromosomal breakpoints. Identifying genes that flank chromosomal reconfigurations is thus essential for cancer cytogenetics. To simplify and expedite this identification, we have developed a novel approach, based on simultaneous spectral karyotyping and fluorescence in situ hybridization (FISH) which, in a single step, can identify gross chromosomal aberrations as well as detect the involvement of specific loci in these rearrangements. Signals for specifically queried genes (FISH probe) were easily detectable in metaphase cells, together with the signals from painted chromosomes (spectral karyotyping probes). The concentration and size of the FISH probes could cover a wide range and still be used successfully. Some of the nucleotide-bound dyes used for the labeling, as Cy3, Spectrum Orange, Alexa 594, Texas Red, and Rhodamine 110, were particularly efficient. More than one gene can be queried in the same metaphase, because multiple FISH probes could be hybridized simultaneously. To demonstrate this technique, we applied it to the myeloma cell line Karpas 620, which has numerous chromosomal rearrangements. The approach that we present here will be particularly useful for the analysis of complex karyotypes and for testing hypotheses arising from cancer gene expression studies. Published 2000 Wiley-Liss, Inc.


Subject(s)
Genes, Neoplasm/genetics , In Situ Hybridization, Fluorescence/methods , Translocation, Genetic/genetics , Chromosome Aberrations/genetics , DNA Probes/metabolism , DNA, Neoplasm/metabolism , Fluorescent Dyes/metabolism , Genetic Markers/genetics , Humans , Karyotyping/methods , Tumor Cells, Cultured
12.
Proc Natl Acad Sci U S A ; 97(1): 228-33, 2000 Jan 04.
Article in English | MEDLINE | ID: mdl-10618400

ABSTRACT

Translocations involving c-myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c-myc or L-myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c-myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c-myc occurs as a late event. All informative MM cell lines show monoallelic expression of c-myc. For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c-myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c-myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.


Subject(s)
Chromosome Aberrations/genetics , Gene Expression Regulation, Neoplastic/genetics , Genes, myc/genetics , Multiple Myeloma/genetics , Chromosome Disorders , Chromosome Painting , Gene Rearrangement , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , In Situ Hybridization, Fluorescence , Karyotyping , RNA, Messenger/genetics , Translocation, Genetic/genetics , Tumor Cells, Cultured
13.
Nature ; 399(6737): 691-4, 1999 Jun 17.
Article in English | MEDLINE | ID: mdl-10385121

ABSTRACT

The establishment of the main body axis and the determination of left-right asymmetry are fundamental aspects of vertebrate embryonic development. A link between these processes has been revealed by the frequent finding of midline defects in humans with left-right anomalies. This association is also seen in a number of mutations in mouse and zebrafish, and in experimentally manipulated Xenopus embryos. However, the severity of laterality defects accompanying abnormal midline development varies, and the molecular basis for this variation is unknown. Here we show that mouse embryos lacking the early-response gene SIL have axial midline defects, a block in midline Sonic hedgehog (Shh) signalling and randomized cardiac looping. Comparison with Shh mutant embryos, which have axial defects but normal cardiac looping, indicates that the consequences of abnormal midline development for left-right patterning depend on the time of onset, duration and severity of disruption of the normal asymmetric patterns of expression of nodal, lefty-2 and Pitx2.


Subject(s)
Body Patterning/genetics , Embryonic and Fetal Development/genetics , Nuclear Proteins , Oncogene Proteins, Fusion , Proteins/genetics , Trans-Activators , Animals , Body Patterning/physiology , Embryo, Mammalian/abnormalities , Embryonic and Fetal Development/physiology , Gene Targeting , Heart/embryology , Hedgehog Proteins , Homeodomain Proteins/biosynthesis , Intracellular Signaling Peptides and Proteins , Left-Right Determination Factors , Mice , Mice, Nude , Mutagenesis , Neural Tube Defects/genetics , Nodal Protein , Paired Box Transcription Factors , Proteins/metabolism , Proteins/physiology , Signal Transduction , Stem Cells , Transcription Factors/biosynthesis , Transforming Growth Factor beta/biosynthesis , Homeobox Protein PITX2
14.
Carcinogenesis ; 20(2): 185-91, 1999 Feb.
Article in English | MEDLINE | ID: mdl-10069452

ABSTRACT

Evidence is accumulating which indicates that cyclooxygenase-2 (COX-2) is involved in the pathogenesis of colorectal cancer. We evaluated the expression of COX-2 in replication error-positive (RER) colon cancers, colon cancers metastatic to liver and azoxymethane (AOM)-induced rat colonic tumors. Immunohistochemistry showed that COX-2 was low to undetectable in normal human mucosa, but abundant in the RER adenocarcinomas we examined. COX-2 immunoreactivity in metastatic colon cancers was less abundant, but clearly detectable. In the colon of AOM-treated rats, COX-2 protein was not detectable in normal mucosa, but present in most of the epithelial cells comprising the tumors. The TGF-beta1 staining pattern in these human and rat tumors was similar to that observed for COX-2. The role of TGF-beta in RER adenocarcinomas is complex because of the increased mutation rate of TGF-beta type II receptors. Northern analysis showed abundant TGF-beta1 mRNA in AOM-induced tumors, but not in paired mucosa. TGF-beta1 induced the expression of COX-2 mRNA and protein in intestinal epithelial cells (IEC-6). Chronic TGF-beta1 treatment caused a TGF-beta-dependent overexpression of COX-2 in rat intestinal epithelial cells (RIE-1). TGF-beta1 may regulate COX-2 expression during the colonic adenoma to carcinoma sequence.


Subject(s)
Colonic Neoplasms/metabolism , Isoenzymes/metabolism , Neoplasm Proteins/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Transforming Growth Factor beta/metabolism , Animals , Azoxymethane , Carcinogens , Colon/metabolism , Colonic Neoplasms/chemically induced , Colonic Neoplasms/pathology , Cyclooxygenase 1 , Cyclooxygenase 2 , Enzyme Induction , Epithelial Cells , Humans , Immunohistochemistry , Intestinal Mucosa/metabolism , Liver Neoplasms/metabolism , Liver Neoplasms/secondary , Male , Membrane Proteins , RNA, Messenger/metabolism , Rats , Rats, Inbred F344 , Transforming Growth Factor beta/pharmacology
15.
Neoplasia ; 1(4): 356-67, 1999 Oct.
Article in English | MEDLINE | ID: mdl-10935491

ABSTRACT

We have developed a "comparative growth assay" that complements current assays of drug effects based on cytotoxicity. A co-culture of two cell lines, one of which is fluorescently labeled, is exposed to a cytotoxic agent and the proportion of fluorescent cells is compared with that of a baseline unexposed co-culture. For demonstration purposes, two HCT116 cell lines (an hMLH1 homozygous and an hMLH1 heterozygous mutant), altered by insertion of vector alone or the same vector carrying an insert for the expression of enhanced green fluorescent protein (EGFP), were exposed to numerous "anti-cancer" agents. The assay was further validated in a system of two cell lines differing only in the expression of the breast cancer resistance protein (BRCP). The assay allowed the estimation of the duration of action of a particular agent. Assessment of the agent's differential activity over a given time in culture could be expressed as a selection rate, which we chose to describe on an "average selection per day" basis. We conclude that this assay: 1) provides insight into the differential dynamic effects of chemotherapeutic agents or radiation; and 2) allows, through the use of matched cell lines, the investigation of critical physiologic features that govern cell sensitivity.


Subject(s)
Cell Culture Techniques/methods , Mutation , Neoplasm Proteins , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/biosynthesis , ATP-Binding Cassette Transporters/genetics , Anti-Bacterial Agents/pharmacology , Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Division/drug effects , Cell Division/radiation effects , Coculture Techniques , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Dose-Response Relationship, Drug , Dose-Response Relationship, Radiation , Drug Resistance, Neoplasm/genetics , Flow Cytometry , Fluorescent Dyes/pharmacology , Genetic Vectors , Gentamicins/pharmacology , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Methylnitronitrosoguanidine/pharmacology , Mitoxantrone/pharmacology , Time Factors , Transfection , Tumor Cells, Cultured
16.
Int Immunol ; 10(10): 1539-49, 1998 Oct.
Article in English | MEDLINE | ID: mdl-9796921

ABSTRACT

In order to dissect the correlation between aberrant TAL1 basic-helix-loop-helix (b-HLH) expression and the exclusive development of T cell acute lymphoblastic leukemias (T-ALL) of the TCRalphabeta lineage, we have assessed the ability of class A b-HLH proteins to regulate the TCRalpha and delta enhancers. We demonstrate that E47S binds to TCRalpha but not to TCRdelta E-boxes in vitro. Despite this, neither E2-5 nor HEB transactivate the TCRalpha enhancer in NIH 3T3, nor did Id1 modify endogenously driven TCRalpha [alphaE1-4] activity in a TCRalphabeta cell line. We also demonstrate that TAL1 inhibits both binding of E47S to aE3 and aE4 and endogenous transactivation of the TCRalpha enhancer. Comparison of the activity of the minimal [alphaE1-2] fragment, which contains no E-boxes, with the accessory [aE3-4] fragment, which contains two, suggested some contribution from the latter to TCRalpha enhancer activity in HPB-ALL. TCR [alphaE1-2] activity was partially (40%) inhibited by TAL1 but not at all by Id1. In contrast, [alphaE3-4] activity was almost completely inhibited by TAL1 (80%) and slightly reduced by Id1 (15%). These data demonstrate that class A b-HLH regulation of the TCRalpha enhancer E-boxes differs from their B lymphoid Igmicro counterparts and suggest a novel mechanism of transcriptional inhibition by TAL1, which may be, at least partly, independent of E-box-mediated activation, as we currently recognize it. They also clearly demonstrate that the restriction of TAL1 deregulation to T-ALL of the TCRalphabeta lineage is not due to induction of TCRalpha enhancer activity by the TAL1 protein.


Subject(s)
DNA-Binding Proteins/metabolism , Helix-Loop-Helix Motifs/physiology , Proto-Oncogene Proteins , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, gamma-delta/genetics , Repressor Proteins , Trans-Activators/metabolism , Transcription Factors/metabolism , 3T3 Cells/metabolism , Animals , Basic Helix-Loop-Helix Transcription Factors , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/pharmacology , Enhancer Elements, Genetic/drug effects , Enhancer Elements, Genetic/physiology , Immunoglobulins/genetics , Inhibitor of Differentiation Protein 1 , Leukemia-Lymphoma, Adult T-Cell/genetics , Mice , Plasmids/genetics , T-Cell Acute Lymphocytic Leukemia Protein 1 , Trans-Activators/pharmacology , Transcription Factors/pharmacology , Transcriptional Activation/drug effects
17.
J Exp Med ; 188(3): 439-50, 1998 Aug 03.
Article in English | MEDLINE | ID: mdl-9687522

ABSTRACT

In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34(+) hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony-stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit-dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL- or dn-SCL- expressing cells. c-kit mRNA, c-kit promoter activity, and the response to SF were rescued by SCL overexpression in the antisense or dn-SCL transfectants. Furthermore, ectopic c-kit expression in as-SCL transfectants is sufficient to restore cell survival in response to SF. Finally, enforced SCL in the pro-B cell line Ba/F3, which is both SCL and c-kit negative is sufficient to induce c-Kit and SF responsiveness. Together, these results indicate that c-kit, a gene that is essential for the survival of primitive hemopoietic cells, is a downstream target of the transcription factor SCL.


Subject(s)
DNA-Binding Proteins/metabolism , Proto-Oncogene Proteins c-kit/biosynthesis , Proto-Oncogene Proteins , Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors , Bone Marrow Cells/metabolism , Cell Survival , DNA-Binding Proteins/genetics , Gene Expression , Granulocyte-Macrophage Colony-Stimulating Factor/pharmacology , Humans , Interleukin-3/pharmacology , Oligonucleotides, Antisense , Proto-Oncogene Proteins c-kit/genetics , Stem Cell Factor/pharmacology , T-Cell Acute Lymphocytic Leukemia Protein 1 , Transcription Factors/genetics , Tumor Cells, Cultured
18.
Cancer Res ; 57(20): 4478-81, 1997 Oct 15.
Article in English | MEDLINE | ID: mdl-9377556

ABSTRACT

Beta-catenin has been identified as an oncogene in colon cancer and melanoma. Phosphorylation of sites in exon 3 of beta-catenin leads to degradation of this protein. These sites are primary targets for activating mutations. The frequency with which oncogenic mutations at these sites are found in colorectal cancer is unknown, as is the frequency of their occurrence in other malignancies. We analyzed 92 colorectal cancers (CRCs) and 57 cancer cell lines (representing a diversity of tumor types) to determine the frequency of activating mutations in this gene. Mutations in exon 3 of beta-catenin were found in 2 of 92 CRCs and in the colorectal cancer cell line HCT 116. Both tumors with beta-catenin mutations exhibited widespread microsatellite instability, which is indicative of a replication error phenotype, a phenotype known to be present in HCT 116. This suggests that mutations in beta-catenin are infrequent in CRC and miscellaneous cancer cell lines and may occur in association with a replication error phenotype.


Subject(s)
Colorectal Neoplasms/genetics , Cytoskeletal Proteins/genetics , DNA Replication/genetics , Microsatellite Repeats , Point Mutation , Trans-Activators , Adult , Aged , Alanine , Amino Acid Substitution , Base Sequence , Cadherins/genetics , Colorectal Neoplasms/pathology , Cytoskeletal Proteins/biosynthesis , Exons , Female , Humans , Male , Middle Aged , Neoplasm Staging , Phenotype , Threonine , Tumor Cells, Cultured , beta Catenin
19.
Cancer Res ; 57(19): 4408-13, 1997 Oct 01.
Article in English | MEDLINE | ID: mdl-9331104

ABSTRACT

Pilot studies in human populations have demonstrated a correlation between the level of antigen receptor trans-rearrangements and risk (at the population level) of lymphoid malignancy. Irradiation of newborn severe combined immune deficiency mice results in an increased risk of subsequent development of thymic lymphoma (100% of mice so irradiated are dead of thymic lymphoma by 20 weeks of age). We, therefore, assayed the occurrence of trans-rearrangements in this well-controlled mouse mutant system and found a 50-100-fold increase in the absolute number of TCRGV-TCRBJ trans-rearrangements compared to unirradiated littermates (and a comparable fold increase over age-matched BALB/c mice) at 2 weeks following irradiation. We also found a marked disproportion in generating trans-rearrangements versus intralocus rearrangements in the severe combined immune deficiency system compared to BALB/c, independent of irradiation. The trans-rearrangements noted were polyclonal in nature. These data, again, suggest that the absolute level of antigen receptor trans-rearrangements may serve as a biomarker of lymphoma risk.


Subject(s)
Gene Rearrangement, beta-Chain T-Cell Antigen Receptor , Gene Rearrangement, gamma-Chain T-Cell Antigen Receptor , Lymphoma/genetics , Neoplasms, Radiation-Induced/genetics , Receptors, Antigen, T-Cell/genetics , Animals , Animals, Newborn , Biomarkers , DNA Nucleotidyltransferases/metabolism , DNA Repair/genetics , Female , Lymphoma/etiology , Male , Mice , Mice, Inbred BALB C , Mice, SCID , Neoplasms, Radiation-Induced/etiology , Risk , Severe Combined Immunodeficiency/complications , Severe Combined Immunodeficiency/genetics , VDJ Recombinases
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