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2.
Nat Genet ; 49(11): 1624-1632, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28945253

ABSTRACT

The G-protein-coupled receptors LGR4, LGR5 and LGR6 are Wnt signaling mediators, but their functions in squamous cell carcinoma (SCC) are unclear. Using lineage tracing in Lgr5-EGFP-CreERT2/Rosa26-Tomato and Lgr6-EGFP-CreERT2/Rosa26-Tomato reporter mice, we demonstrate that Lgr6, but not Lgr5, acts as an epithelial stem cell marker in SCCs in vivo. We identify, by single-molecule in situ hybridization and cell sorting, rare cells positive for Lgr6 expression in immortalized keratinocytes and show that their frequency increases in advanced SCCs. Lgr6 expression is enriched in cells with stem cell characteristics, and Lgr6 downregulation in vivo causes increased epidermal proliferation with expanded lineage tracing from epidermal stem cells positive for Lgr6 expression. Surprisingly, mice with germline knockout of Lgr6 are predisposed to SCC development, through a mechanism that includes compensatory upregulation of Lgr5. These data provide a model for human patients with germline loss-of-function mutations in Wnt pathway genes, including RSPO1 or LGR4, who show increased susceptibility to squamous tumor development.


Subject(s)
Carcinoma, Squamous Cell/genetics , Gene Expression Regulation, Neoplastic , Keratinocytes/metabolism , Neoplastic Stem Cells/metabolism , Receptors, G-Protein-Coupled/genetics , Skin Neoplasms/genetics , Animals , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/pathology , Cell Line, Transformed , Epidermis/metabolism , Epidermis/pathology , Humans , Keratinocytes/pathology , Mice , Mice, Transgenic , Neoplastic Stem Cells/pathology , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Thrombospondins/genetics , Thrombospondins/metabolism
3.
Stem Cells ; 31(9): 1910-20, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23712803

ABSTRACT

Specification of the cellular hierarchy in the mammary gland involves complex signaling that remains poorly defined. Polycomb group proteins are known to contribute to the maintenance of stem cell identity through epigenetic modifications, leading to stable alterations in gene expression. The polycomb protein family member EZH2 is known to be important for stem cell maintenance in multiple tissues, but its role in mammary gland development and differentiation remains unknown. Our analyses show that EZH2 is predominantly expressed in luminal cells of the mouse mammary epithelium. As mammary gland development occurs mostly after birth, the analysis of EZH2 gene function in postnatal development is precluded by embryonic lethality of conventional EZH2 knockout mice. To investigate the role of EZH2 in normal mammary gland epithelium, we have generated novel transgenic mice that express doxycycline-regulatable short hairpin (sh) RNAs directed against Ezh2. Knockdown of EZH2 results in delayed outgrowth of the mammary epithelium during puberty, due to impaired terminal end bud formation and ductal elongation. Furthermore, our results demonstrate that EZH2 is required to maintain the luminal cell pool and may limit differentiation of luminal progenitors into CD61(+) differentiated luminal cells, suggesting a role for EZH2 in mammary luminal cell fate determination. Consistent with this, EZH2 knockdown reduced lobuloalveolar expansion during pregnancy, suggesting EZH2 is required for the differentiation of luminal progenitors to alveolar cells.


Subject(s)
Mammary Glands, Animal/growth & development , Mammary Glands, Animal/metabolism , Morphogenesis , Polycomb Repressive Complex 2/genetics , Stem Cells/cytology , Stem Cells/metabolism , Animals , Enhancer of Zeste Homolog 2 Protein , Female , Gene Knockdown Techniques , Mammary Glands, Animal/cytology , Mice , Mice, Inbred C57BL , Polycomb Repressive Complex 2/metabolism , Pregnancy , RNA Interference , Reproducibility of Results
4.
Mol Pharmacol ; 81(1): 63-72, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21989258

ABSTRACT

The highly polymorphic human cytochrome P450 2D6 enzyme is involved in the metabolism of up to 25% of all marketed drugs and accounts for significant individual differences in response to CYP2D6 substrates. Because of the differences in the multiplicity and substrate specificity of CYP2D family members among species, it is difficult to predict pathways of human CYP2D6-dependent drug metabolism on the basis of animal studies. To create animal models that reflect the human situation more closely and that allow an in vivo assessment of the consequences of differential CYP2D6 drug metabolism, we have developed a novel straightforward approach to delete the entire murine Cyp2d gene cluster and replace it with allelic variants of human CYP2D6. By using this approach, we have generated mouse lines expressing the two frequent human protein isoforms CYP2D6.1 and CYP2D6.2 and an as yet undescribed variant of this enzyme, as well as a Cyp2d cluster knockout mouse. We demonstrate that the various transgenic mouse lines cover a wide spectrum of different human CYP2D6 metabolizer phenotypes. The novel humanization strategy described here provides a robust approach for the expression of different CYP2D6 allelic variants in transgenic mice and thus can help to evaluate potential CYP2D6-dependent interindividual differences in drug response in the context of personalized medicine.


Subject(s)
Cytochrome P-450 CYP2D6/metabolism , Embryonic Stem Cells/metabolism , Models, Animal , Alleles , Animals , Cell Line , Drug Interactions/physiology , Embryonic Stem Cells/enzymology , Genetic Variation , Humans , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic
5.
Genes Dev ; 24(12): 1281-94, 2010 Jun 15.
Article in English | MEDLINE | ID: mdl-20551174

ABSTRACT

The Myc protein suppresses the transcription of several cyclin-dependent kinase inhibitors (CKIs) via binding to Miz1; whether this interaction is important for Myc's ability to induce or maintain tumorigenesis is not known. Here we show that the oncogenic potential of a point mutant of Myc (MycV394D) that is selectively deficient in binding to Miz1 is greatly attenuated. Binding of Myc to Miz1 is continuously required to repress CKI expression and inhibit accumulation of trimethylated histone H3 at Lys 9 (H3K9triMe), a hallmark of cellular senescence, in T-cell lymphomas. Lymphomas that arise express high amounts of transforming growth factor beta-2 (TGFbeta-2) and TGFbeta-3. Upon Myc suppression, TGFbeta signaling is required to induce CKI expression and cellular senescence and suppress tumor recurrence. Binding of Myc to Miz1 is required to antagonize growth suppression and induction of senescence by TGFbeta. We demonstrate that, since lymphomas express high levels of TGFbeta, they are poised to elicit an autocrine program of senescence upon Myc inactivation, demonstrating that TGFbeta is a key factor that establishes oncogene addiction of T-cell lymphomas.


Subject(s)
Autocrine Communication/physiology , Lymphoma, T-Cell/physiopathology , Nuclear Proteins/metabolism , Protein Inhibitors of Activated STAT/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Transforming Growth Factor beta/metabolism , Animals , Doxycycline/pharmacology , Gene Expression Regulation, Neoplastic/drug effects , HeLa Cells , Humans , Mice , Mice, Transgenic , Mutation/genetics , Proto-Oncogene Proteins c-myc/genetics , Ubiquitin-Protein Ligases
6.
Biotechnol Appl Biochem ; 53(Pt 1): 31-7, 2009 May.
Article in English | MEDLINE | ID: mdl-19032154

ABSTRACT

Antibody-producing Chinese-hamster ovary cells (CHO-DG44) were converted into cells producing antibodies with strongly enhanced ADCC (antibody-dependent cellular cytotoxicity) by knocking down FuT8 (alpha-1,6-fucosyltransferase or fucosyltransferase 8) via constitutive expression of shRNA (short-hairpin RNA) against FuT8. After the introduction of a FuT8 shRNA expression plasmid under the control of a U6 promoter, CHO-DG44 clones with less than 5% residual FuT8 mRNA expression were isolated by selection for neomycin resistance, followed by low affinity nerve growth factor receptor enrichment and selection for LCA [Lens culinaris (culinary lentil) agglutinin] resistance. The CHO-DG44 clones identified produced highly afucosylated anti-[IGF-1R (insulin-like-growth-factor-1 receptor)] antibodies (up to 88%) that exhibited considerably enhanced ADCC compared with anti-IGF-1R wild-type antibodies produced by parental CHO cells. At the same time, antibody productivity was not significantly decreased. Analysis of stability showed that the clones obtained may be suitable for up-scaling, since low residual levels of FuT8 mRNA and production of afucosylated antibodies were maintained for at least 4 weeks.


Subject(s)
Antibodies/immunology , Antibody-Dependent Cell Cytotoxicity , Fucosyltransferases/genetics , RNA Interference , RNA, Small Interfering , Receptor, IGF Type 1/immunology , Animals , Antibody Formation , CHO Cells , Cloning, Molecular , Cricetinae , Cricetulus , Fucosyltransferases/metabolism , Gene Expression , Receptor, IGF Type 1/metabolism , Recombinant Proteins/immunology , Recombinant Proteins/metabolism
7.
Mol Cell Biol ; 23(21): 7648-57, 2003 Nov.
Article in English | MEDLINE | ID: mdl-14560010

ABSTRACT

Miz1 is a member of the POZ domain/zinc finger transcription factor family. In vivo, Miz1 forms a complex with the Myc oncoprotein and recruits Myc to core promoter elements. Myc represses transcription through Miz1 binding sites. We now show that the Miz1 gene is ubiquitously expressed during mouse embryogenesis. In order to elucidate the physiological function of Miz1, we have deleted the mouse Miz1 gene by homologous recombination. Miz1(+/-) mice are indistinguishable from wild-type animals; in contrast, Miz1(-/-) embryos are not viable. They are severely retarded in early embryonic development and do not undergo normal gastrulation. Expression of Goosecoid and Brachyury is detectable in Miz1(-/-) embryos, suggesting that Miz1 is not required for signal transduction by Nodal. Expression of p21Cip1, a target gene of Miz1 is unaltered; in contrast, expression of p57Kip2, another target gene of Miz1 is absent in Miz1(-/-) embryos. Miz1(-/-) embryos succumb to massive apoptosis of ectodermal cells around day 7.5 of embryonic development. Our results show that Miz1 is required for early embryonic development during gastrulation.


Subject(s)
DNA-Binding Proteins/metabolism , Fetal Proteins , Gastrula/physiology , Gene Expression Regulation, Developmental , Morphogenesis , Repressor Proteins , Transcription Factors , Tumor Suppressor Proteins , Animals , Apoptosis/physiology , Cell Cycle Proteins/metabolism , Cell Division/physiology , Cyclin-Dependent Kinase Inhibitor p15 , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p57 , Cyclins/metabolism , DNA-Binding Proteins/genetics , Enzyme Inhibitors/metabolism , Female , Gastrula/cytology , Gene Targeting , Goosecoid Protein , Homeodomain Proteins/metabolism , Humans , In Situ Hybridization , Kruppel-Like Transcription Factors , Mesoderm/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/metabolism , Phenotype , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , T-Box Domain Proteins/metabolism , Transcription, Genetic
8.
Oncogene ; 22(3): 351-60, 2003 Jan 23.
Article in English | MEDLINE | ID: mdl-12545156

ABSTRACT

Inhibition of cellular differentiation is one of the well-known biological activities of c-Myc-family proteins. We show here that Myc represses differentiation-induced expression of the cyclin-dependent kinase (CDK) inhibitor p21CIP1 (CDKN1A, p21), known to play an important role in cell fate decisions during growth and differentiation, in hematopoietic cells. Our results demonstrate that the c-Myc-responsive region is situated in the p21 core promoter. c-Myc binds to this region in vitro and in vivo through interaction with the initiator-binding Zn-finger transcription factor Miz-1, which associates directly with the promoter. Association of Myc with the promoter in vivo correlates inversely with p21 expression. Using mutants of c-Myc with impaired binding to Miz-1, our results further show that repression of p21 promoter/reporters as well as the endogenous p21 gene by Myc depends on interaction with Miz-1. Expression of Miz-1 increases during hematopoietic differentiation and Miz-1 activates the p21 promoter under conditions of low Myc levels, indicating a positive role for free Miz-1 in this process. In conclusion, repression of differentiation-induced p21 expression through Miz-1 may be an important mechanism by which Myc blocks differentiation.


Subject(s)
Cell Differentiation/genetics , Cyclins/genetics , DNA-Binding Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Transcription Factors/metabolism , Base Sequence , Cell Differentiation/drug effects , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/metabolism , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/genetics , Helix-Loop-Helix Motifs , Humans , Kruppel-Like Transcription Factors , Molecular Sequence Data , Mutation , Promoter Regions, Genetic , Protein Structure, Tertiary , Proto-Oncogene Proteins c-myc/genetics , Tetradecanoylphorbol Acetate/pharmacology , Transcription Factors/drug effects , Transcription Factors/genetics , Transcription, Genetic , U937 Cells/drug effects
9.
Mol Cell ; 10(3): 509-21, 2002 Sep.
Article in English | MEDLINE | ID: mdl-12408820

ABSTRACT

The Myc oncoprotein represses initiator-dependent transcription through the POZ domain transcription factor Miz-1. We now show that transactivation by Miz-1 is negatively regulated by association with topoisomerase II binding protein (TopBP1); UV irradiation downregulates expression of TopBP1 and releases Miz-1. Miz-1 binds to the p21Cip1 core promoter in vivo and is required for upregulation of p21Cip1 upon UV irradiation. Using both c-myc(-/-) cells and a point mutant of Myc that is deficient in Miz-1 dependent repression, we show that Myc negatively regulates transcription of p21Cip1 upon UV irradiation and facilitates recovery from UV-induced cell cycle arrest through binding to Miz-1. Our data implicate Miz-1 in a pathway that regulates cell proliferation in response to UV irradiation.


Subject(s)
DNA-Binding Proteins/metabolism , Proto-Oncogene Proteins c-myc/metabolism , Transcription, Genetic/radiation effects , Transcriptional Activation , Tumor Suppressor Proteins , Ultraviolet Rays , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins/metabolism , Cell Division/physiology , Cell Division/radiation effects , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p15 , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclins/genetics , Cyclins/metabolism , Genes, Reporter , Humans , Keratinocytes/cytology , Keratinocytes/physiology , Keratinocytes/radiation effects , Kruppel-Like Transcription Factors , Models, Molecular , Molecular Sequence Data , Nuclear Proteins , Protein Structure, Tertiary , Proto-Oncogene Proteins c-myc/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Transcription Factors , Two-Hybrid System Techniques
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