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1.
Brain Struct Funct ; 220(4): 1971-82, 2015 Jul.
Article in English | MEDLINE | ID: mdl-24744148

ABSTRACT

Tomosyn-1 (STXBP5) is a soluble NSF attachment protein receptor complex-binding protein that inhibits vesicle fusion, but the role of tomosyn-2 (STXBP5L) in the mammalian nervous system is still unclear. Here we generated tomosyn-2 null (Tom2(KO/KO)) mice, which showed impaired motor performance. This was accompanied by synaptic changes at the neuromuscular junction, including enhanced spontaneous acetylcholine release frequency and faster depression of muscle motor endplate potentials during repetitive stimulation. The postsynaptic geometric arrangement and function of acetylcholine receptors were normal. We conclude that tomosyn-2 supports motor performance by regulation of transmitter release willingness to sustain synaptic strength during high-frequency transmission, which makes this gene a candidate for involvement in neuromuscular disorders.


Subject(s)
Motor Activity/genetics , Motor Endplate/metabolism , Neuromuscular Junction/cytology , R-SNARE Proteins/deficiency , Synaptic Transmission/physiology , Adaptor Proteins, Vesicular Transport , Animals , Biophysics , Diaphragm/physiology , Electric Stimulation , Embryo, Mammalian , Gene Expression Regulation/genetics , Hippocampus/cytology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Patch-Clamp Techniques , Psychomotor Performance/physiology , R-SNARE Proteins/genetics , Receptors, Cholinergic/metabolism , Statistics, Nonparametric , Synaptic Potentials/genetics
2.
Carcinogenesis ; 34(11): 2629-38, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23764752

ABSTRACT

Whereas aberrant activation of canonical Wnt/ß-catenin signaling underlies the majority of colorectal cancer cases, the contribution of non-canonical Wnt signaling is unclear. As enhanced expression of the most extensively studied non-canonical Wnt ligand WNT5A is observed in various diseases including colon cancer, WNT5A is gaining attention nowadays. Numerous in vitro studies suggest modulating capacities of WNT5A on proliferation, differentiation, migration and invasion, affecting tumor and non-mutant cells. However, a possible contribution of WNT5A to colorectal cancer remains to be elucidated. We have analyzed WNT5A expression in colorectal cancer profiling data sets, altered WNT5A expression in colon cancer cells and used our inducible Wnt5a transgenic mouse model to gain more insight into the role of WNT5A in intestinal cancer. We observed that increased WNT5A expression is associated with poor prognosis of colorectal cancer patients. WNT5A knockdown in human colon cancer cells caused reduced directional migration, deregulated focal adhesion site formation and reduced invasion, whereas Wnt5a administration promoted the directional migration of colon cancer cells. Despite these observed protumorigenic activities of WNT5A, the induction of Wnt5a expression in intestinal tumors of Apc1638N mice was not sufficient to augment malignancy or metastasis by itself. In conclusion, WNT5A promotes adhesion sites to form in a focal fashion and promotes the directional migration and invasion of colon cancer cells. Although these activities appear insufficient by themselves to augment malignancy or metastasis in Apc1638N mice, they might explain the poor colon cancer prognosis associated with enhanced WNT5A expression.


Subject(s)
Adenomatous Polyposis Coli Protein/physiology , Cell Movement , Cell Proliferation , Cell Transformation, Neoplastic/pathology , Colonic Neoplasms/pathology , Intestines/pathology , Proto-Oncogene Proteins/metabolism , Wnt Proteins/metabolism , Animals , Apoptosis , Blotting, Western , Cell Adhesion , Cell Transformation, Neoplastic/metabolism , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Focal Adhesions , Humans , Immunoenzyme Techniques , Intestinal Mucosa/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Neoplasm Invasiveness , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Wnt Proteins/genetics , Wnt-5a Protein
3.
Dev Biol ; 369(1): 91-100, 2012 Sep 01.
Article in English | MEDLINE | ID: mdl-22691362

ABSTRACT

Wnt5a is essential during embryonic development, as indicated by mouse Wnt5a knockout embryos displaying outgrowth defects of multiple structures including the gut. The dynamics of Wnt5a involvement in these processes is unclear, and perinatal lethality of Wnt5a knockout embryos has hampered investigation of Wnt5a during postnatal stages in vivo. Although in vitro studies have suggested a relevant role for Wnt5a postnatally, solid evidence for a significant impact of Wnt5a within the complexity of an adult organism is lacking. We generated a tightly-regulated inducible Wnt5a transgenic mouse model and investigated the effects of Wnt5a induction during different time-frames of embryonic development and in adult mice, focusing on the gastrointestinal tract. When induced in embryos from 10.5 dpc onwards, Wnt5a expression led to severe outgrowth defects affecting the gastrointestinal tracts, limbs, facial structures and tails, closely resembling the defects observed in Wnt5a knockout mice. However, Wnt5a induction from 13.5 dpc onwards did not cause this phenotype, indicating that the most critical period for Wnt5a in embryonic development is prior to 13.5 dpc. In adult mice, induced Wnt5a expression did not reveal abnormalities, providing the first in vivo evidence that Wnt5a has no major impact on mouse intestinal homeostasis postnatally. Protein expression of Wnt5a receptor Ror2 was strongly reduced in adult intestine compared to embryonic stages. Moreover, we uncovered a regulatory process where induction of Wnt5a causes downregulation of its receptor Ror2. Taken together, our results indicate a role for Wnt5a during a restricted time-frame of embryonic development, but suggest no impact during homeostatic postnatal stages.


Subject(s)
Aging/genetics , Embryo, Mammalian/metabolism , Embryonic Development/genetics , Intestines/embryology , Wnt Proteins/metabolism , Aging/drug effects , Animals , Cell Lineage/drug effects , Cell Lineage/genetics , Down-Regulation/drug effects , Down-Regulation/genetics , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryonic Development/drug effects , Gene Expression Regulation, Developmental/drug effects , Intestinal Mucosa/metabolism , Intestines/cytology , Intestines/drug effects , Mice , Mice, Transgenic , Models, Animal , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Tetracycline/pharmacology , Wnt Proteins/genetics , Wnt-5a Protein
4.
DNA Repair (Amst) ; 10(12): 1252-61, 2011 Dec 10.
Article in English | MEDLINE | ID: mdl-22036606

ABSTRACT

Fanconi anemia (FA) is a heritable disease characterized by bone marrow failure, congenital abnormalities, and cancer predisposition. The 15 identified FA genes operate in a molecular pathway to preserve genomic integrity. Within this pathway the FA core complex operates as an ubiquitin ligase that activates the complex of FANCD2 and FANCI to coordinate DNA repair. The FA core complex is formed by at least 12 proteins. However, only the FANCL subunit displays ubiquitin ligase activity. FANCA and FANCG are members of the FA core complex for which no other functions have been described than to participate in protein interactions. In this study we generated mice with combined null alleles for Fanca and Fancg to identify extended functions for these genes by characterizing the double mutant mice and cells. Double mutant a(-/-)/g(-/-) mice were born at near Mendelian frequencies without apparent developmental abnormalities. Histological analysis of a(-/-)/g(-/-) mice revealed a Leydig cell hyperplasia and frequent vacuolization of Sertoli cells in testes, while ovaries were depleted from developing follicles and displayed an interstitial cell hyperplasia. These gonadal aberrations were associated with a compromised fertility of a(-/-)/g(-/-) males and females. During the first year of life a(-/-)/g(-/-) did not develop malignancies or bone marrow failure. At the cellular level a(-/-)/g(-/-), Fanca(-/-), and Fancg(-/-) cells proved equally compromised in DNA crosslink and homology-directed repair. Overall the phenotype of a(-/-)/g(-/-) double knockout mice and cells appeared highly similar to the phenotype of Fanca or Fancg single knockouts. The lack of an augmented phenotype suggest that null mutations in Fanca or Fancg are fully epistatic, making additional important functions outside of the FA core complex highly unlikely.


Subject(s)
Epistasis, Genetic/genetics , Fanconi Anemia Complementation Group A Protein/genetics , Fanconi Anemia Complementation Group G Protein/genetics , Fanconi Anemia/genetics , Mutation/genetics , Active Transport, Cell Nucleus/drug effects , Animals , Bone Marrow Cells/cytology , Cell Line , Cell Nucleus/drug effects , Cell Nucleus/metabolism , Chromosome Breakage/drug effects , Embryo, Mammalian , Female , Fertility/genetics , Fibroblasts/cytology , Fluorobenzenes/pharmacology , Hematologic Tests , Humans , Male , Mice , Ovary/metabolism , Phthalazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , Stem Cells/drug effects , Stem Cells/metabolism , Testis/metabolism
5.
Gut ; 60(9): 1204-12, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21307168

ABSTRACT

Objective Deregulation of the Wnt signalling pathway by mutations in the Apc or ß-catenin genes underlies colorectal carcinogenesis. As a result, ß-catenin stabilises, translocates to the nucleus, and activates gene transcription. Intestinal tumours show a heterogeneous pattern of nuclear ß-catenin, with the highest levels observed at the invasion front. Activation of receptor tyrosine kinases in these tumour areas by growth factors expressed by surrounding stromal cells phosphorylate ß-catenin at tyrosine residues, which is thought to increase ß-catenin nuclear translocation and tumour invasiveness. This study investigates the relevance of ß-catenin tyrosine phosphorylation for Wnt signalling and intestinal tumorigenesis in vivo. Design A conditional knock-in mouse model was generated into which the phospho-mimicking Y654E modification in the endogenous ß-catenin gene was introduced. Results This study provided in vivo evidence that ß-catenin(E654) is characterised by reduced affinity for cadherins, increased signalling and strongly increased phosphorylation at serine 675 by protein kinase A (PKA). In addition, homozygosity for the ß-catenin(E654) targeted allele caused embryonic lethality, whereas heterozygosity predisposed to intestinal tumour development, and strongly enhanced Apc-driven intestinal tumour initiation associated with increased nuclear accumulation of ßcatenin. Surprisingly, the expression of ß-catenin(E654) did not affect histological grade or induce tumour invasiveness. Conclusions A thus far unknown mechanism was uncovered in which Y654 phosphorylation of ß-catenin facilitates additional phosphorylation at serine 675 by PKA. In addition, in contrast to the current belief that ß-catenin Y654 phosphorylation increases tumour progression to a more invasive phenotype, these results show that it rather increases tumour initiation by enhancing Wnt signalling.


Subject(s)
Cell Transformation, Neoplastic/metabolism , Colorectal Neoplasms/metabolism , Wnt Proteins/physiology , beta Catenin/metabolism , Adenoma/genetics , Adenoma/metabolism , Animals , COS Cells , Cadherins/metabolism , Cell Membrane/metabolism , Cell Transformation, Neoplastic/genetics , Chlorocebus aethiops , Colorectal Neoplasms/genetics , Cyclic AMP-Dependent Protein Kinases/pharmacology , Embryo Loss/genetics , Gene Knock-In Techniques , Genes, APC , Genotype , Heterozygote , Homozygote , Mice , Mice, Inbred C57BL , Phosphorylation/drug effects , Phosphorylation/physiology
6.
FASEB J ; 25(1): 280-9, 2011 Jan.
Article in English | MEDLINE | ID: mdl-20876209

ABSTRACT

For amphiphilic anticancer drugs, such as the anthracyclin doxorubicin (Dox), uptake by tumor cells involves slow diffusion across the plasma membrane, a limiting factor in clinical oncology. Previously, we discovered that preinsertion of short-chain sphingolipids such as N-octanoyl-glucosylceramide (GC) in the tumor cell membrane enhances cellular Dox uptake. In the present study, we apply this strategy in vitro and in vivo by coadministering GC and Dox in a lipid nanovesicle (LNV). GC enrichment of Dox-LNVs strongly enhanced in vitro cytotoxicity toward B16 melanoma and A431 carcinoma, as evidenced by 6-fold decreased IC(50) values compared with Dox-LNVs. This correlated with enhanced cellular Dox uptake observed by confocal microscopy. Intravital optical imaging in window chamber-bearing mice with orthotopically implanted B16 melanoma demonstrated enhanced GC-mediated Dox delivery to tumor cells. Treatment of nude mice bearing human A431 xenografts with 6 mg/kg GC-Dox-LNVs almost doubled the tumor growth delay compared with Dox-LNVs. A second administration of 5 mg/kg after 3 d induced even 3-fold delay in tumor growth, while no systemic toxicity was found. GC-enriched Dox-LNVs displayed superior in vitro and in vivo antitumor activity, without systemic toxicity. This new drug delivery concept, aiming at increased membrane permeability for amphiphilic drugs, provides an opportunity to improve cancer chemotherapy.


Subject(s)
Doxorubicin/pharmacology , Glucosylceramides/chemistry , Nanostructures/chemistry , Neoplasms/drug therapy , Animals , Antibiotics, Antineoplastic/administration & dosage , Antibiotics, Antineoplastic/chemistry , Antibiotics, Antineoplastic/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Dose-Response Relationship, Drug , Doxorubicin/administration & dosage , Doxorubicin/chemistry , Drug Delivery Systems/methods , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Microscopy, Confocal , Neoplasms/pathology , Treatment Outcome , Unilamellar Liposomes/chemistry , Xenograft Model Antitumor Assays
7.
Proc Natl Acad Sci U S A ; 106(51): 21836-41, 2009 Dec 22.
Article in English | MEDLINE | ID: mdl-20007784

ABSTRACT

The induction of skin cancer involves both mutagenic and proliferative responses of the epidermis to ultraviolet (UV) light. It is believed that tumor initiation requires the mutagenic replication of damaged DNA by translesion synthesis (TLS) pathways. The mechanistic basis for the induction of proliferation, providing tumor promotion, is poorly understood. Here, we have investigated the role of TLS in the initiation and promotion of skin carcinogenesis, using a sensitive nucleotide excision repair-deficient mouse model that carries a hypomorphic allele of the error-prone TLS gene Rev1. Despite a defect in UV-induced mutagenesis, skin carcinogenesis was accelerated in these mice. This paradoxical phenotype was caused by the induction of inflammatory hyperplasia of the mutant skin that provides strong tumor promotion. The induction of hyperplasia was associated with mild and transient replicational stress of the UV-damaged genome, triggering DNA damage signaling and senescence. The concomitant expression of Interleukin-6 (IL-6) is in agreement with an executive role for IL-6 and possibly other cytokines in the autocrine induction of senescence and the paracrine induction of inflammatory hyperplasia. In conclusion, error-prone TLS suppresses tumor-promoting activities of UV light, thereby controlling skin carcinogenesis.


Subject(s)
DNA Damage , DNA Replication , Alleles , Animals , DNA Repair , Interleukin-6/genetics , Interleukin-6/physiology , Mice , Models, Animal , Neoplasms, Radiation-Induced/genetics , Skin Neoplasms/genetics , Ultraviolet Rays
8.
BMC Dev Biol ; 9: 26, 2009 Apr 08.
Article in English | MEDLINE | ID: mdl-19356224

ABSTRACT

BACKGROUND: During skeletogenesis, protein levels of beta-catenin in the canonical Wnt signaling pathway determine lineage commitment of skeletal precursor cells to osteoblasts and chondrocytes. Adenomatous polyposis coli (Apc) is a key controller of beta-catenin turnover by down-regulating intracellular levels of beta-catenin. RESULTS: To investigate whether Apc is involved in lineage commitment of skeletal precursor cells, we generated conditional knockout mice lacking functional Apc in Col2a1-expressing cells. In contrast to other models in which an oncogenic variant of beta-catenin was used, our approach resulted in the accumulation of wild type beta-catenin protein due to functional loss of Apc. Conditional homozygous Apc mutant mice died perinatally showing greatly impaired skeletogenesis. All endochondral bones were misshaped and lacked structural integrity. Lack of functional Apc resulted in a pleiotropic skeletal cell phenotype. The majority of the precursor cells lacking Apc failed to differentiate into chondrocytes or osteoblasts. However, skeletal precursor cells in the proximal ribs were able to escape the noxious effect of functional loss of Apc resulting in formation of highly active osteoblasts. Inactivation of Apc in chondrocytes was associated with dedifferentiation of these cells. CONCLUSION: Our data indicate that a tight Apc-mediated control of beta-catenin levels is essential for differentiation of skeletal precursors as well as for the maintenance of a chondrocytic phenotype in a spatio-temporal regulated manner.


Subject(s)
Adenomatous Polyposis Coli Protein/genetics , Bone and Bones/metabolism , Embryo, Mammalian/metabolism , beta Catenin/genetics , Animals , Bone and Bones/cytology , Bone and Bones/embryology , Cell Differentiation/genetics , Chondrocytes/cytology , Chondrocytes/metabolism , Chondrogenesis/genetics , Collagen Type II/genetics , Embryo, Mammalian/embryology , Gene Expression Regulation, Developmental , In Situ Hybridization , Mice , Mice, Knockout , Mice, Transgenic , Osteoblasts/cytology , Osteoblasts/metabolism , Osteogenesis/genetics , Phenotype , Time Factors
9.
J Clin Invest ; 117(11): 3583-92, 2007 Nov.
Article in English | MEDLINE | ID: mdl-17975676

ABSTRACT

Cytochrome P450 3A (CYP3A) enzymes constitute an important detoxification system that contributes to primary metabolism of more than half of all prescribed medications. To investigate the physiological and pharmacological roles of CYP3A, we generated Cyp3a-knockout (Cyp3a-/-) mice lacking all functional Cyp3a genes. Cyp3a-/- mice were viable, fertile, and without marked physiological abnormalities. However, these mice exhibited severely impaired detoxification capacity when exposed to the chemotherapeutic agent docetaxel, displaying higher exposure levels in response to both oral and intravenous administration. These mice also demonstrated increased sensitivity to docetaxel toxicity, suggesting a primary role for Cyp3a in xenobiotic detoxification. To determine the relative importance of intestinal versus hepatic Cyp3a in first-pass metabolism, we generated transgenic Cyp3a-/- mice expressing human CYP3A4 in either the intestine or the liver. Expression of CYP3A4 in the intestine dramatically decreased absorption of docetaxel into the bloodstream, while hepatic expression aided systemic docetaxel clearance. These results suggest that CYP3A expression determines impairment of drug absorption and efficient systemic clearance in a tissue-specific manner. The genetic models used in this study provide powerful tools to further study CYP3A-mediated xenobiotic metabolism, as well as interactions between CYP3A and other detoxification systems.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Mice, Knockout , Xenobiotics/metabolism , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Biological Availability , Cytochrome P-450 CYP3A , Cytochrome P-450 Enzyme System/genetics , Docetaxel , Humans , Mice , Mice, Transgenic , Microsomes/metabolism , Phenotype , Taxoids/administration & dosage , Taxoids/pharmacokinetics
10.
Cancer Res ; 66(6): 3278-86, 2006 Mar 15.
Article in English | MEDLINE | ID: mdl-16540681

ABSTRACT

Cancer immunotherapy based on vaccination with defined tumor antigens has not yet shown strong clinical efficacy, despite promising results in preclinical models. This discrepancy might result from the fact that available preclinical models rely on transplantable tumors, which do not recapitulate the long-term host-tumor interplay that occurs in patients during progressive tumor development and results in tumor tolerance. To create a faithful preclinical model for cancer immunotherapy, we generated a transgenic mouse strain developing autologous melanomas expressing a defined tumor antigen recognized by T cells. We chose the antigen encoded by P1A, a well-characterized murine cancer germ line gene. To transform melanocytes, we aimed at simultaneously activating the Ras pathway and inactivating tumor suppressor Ink4a/Arf, thereby reproducing two genetic events frequently observed in human melanoma. The melanomas are induced by s.c. injection of 4-OH-tamoxifen (OHT). By activating a CreER recombinase expressed from a melanocyte-specific promoter, this treatment induces the loss of the conditional Ink4a/Arf gene in melanocytes. Because the CreER gene itself is also flanked by loxP sites, the activation of CreER also induces the deletion of its own coding sequence and thereby allows melanocyte-specific expression of genes H-ras and P1A, which are located downstream on the same transgene. All melanomas induced in those mice with OHT show activation of the Ras pathway and deletion of gene Ink4a/Arf. In addition, these melanomas express P1A and are recognized by P1A-specific T lymphocytes. This model will allow to characterize the interactions between the immune system and naturally occurring tumors and thereby to optimize immunotherapy approaches targeting a defined tumor antigen.


Subject(s)
Antigens, Neoplasm/biosynthesis , Melanoma, Experimental/immunology , Animals , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Cyclin-Dependent Kinase Inhibitor p16/antagonists & inhibitors , Cyclin-Dependent Kinase Inhibitor p16/biosynthesis , Cyclin-Dependent Kinase Inhibitor p16/genetics , Disease Models, Animal , Melanoma, Experimental/genetics , Mice , Mice, Transgenic , Recombination, Genetic , Tamoxifen/analogs & derivatives , Tumor Suppressor Protein p14ARF/antagonists & inhibitors , Tumor Suppressor Protein p14ARF/biosynthesis , Tumor Suppressor Protein p14ARF/genetics , ras Proteins/genetics
11.
Nat Immunol ; 4(1): 49-54, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12469117

ABSTRACT

It has been proposed that HIV-1, in addition to directly infecting and killing CD4+ T cells, causes T cell dysfunction and T cell loss by chronic immune activation. We analyzed the effects of chronic immune activation in mice that constitutively expressed CD70, the ligand for the tumor necrosis factor receptor family member CD27, on B cells. CD70 transgenic (CD70 Tg) mice showed a progressive conversion of naive T cells into effector-memory cells, which culminated in the depletion of naive T cells from lymph nodes and spleen. T cell changes depended on continuous CD27-CD70 interactions and T cell antigen receptor stimulation. Despite this hyperactive immune system, CD70 Tg mice died aged 6-8 months from Pneumocystis carinii infection, a hallmark of T cell immunodeficiency. Thus, persistent delivery of costimulatory signals via CD27-CD70 interactions, as may occur during chronic active viral infections, can exhaust the T cell pool and is sufficient to induce lethal immunodeficiency.


Subject(s)
Antigens, CD , Immunologic Deficiency Syndromes/etiology , Membrane Proteins/metabolism , T-Lymphocytes/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 7/metabolism , Animals , CD27 Ligand , Cytopathogenic Effect, Viral , HIV Infections/etiology , HIV Infections/immunology , HIV-1/immunology , HIV-1/pathogenicity , Humans , Immunologic Deficiency Syndromes/genetics , Immunologic Deficiency Syndromes/immunology , Immunologic Memory , Lymphocyte Activation , Lymphocyte Count , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Pneumonia, Pneumocystis/etiology , Pneumonia, Pneumocystis/immunology , T-Lymphocytes/pathology
12.
Proc Natl Acad Sci U S A ; 99(24): 15649-54, 2002 Nov 26.
Article in English | MEDLINE | ID: mdl-12429862

ABSTRACT

The breast cancer resistance protein (BCRPABCG2) is a member of the ATP-binding cassette family of drug transporters and confers resistance to various anticancer drugs. We show here that mice lacking Bcrp1Abcg2 become extremely sensitive to the dietary chlorophyll-breakdown product pheophorbide a, resulting in severe, sometimes lethal phototoxic lesions on light-exposed skin. Pheophorbide a occurs in various plant-derived foods and food supplements. Bcrp1 transports pheophorbide a and is highly efficient in limiting its uptake from ingested food. Bcrp1(-/-) mice also displayed a previously unknown type of protoporphyria. Erythrocyte levels of the heme precursor and phototoxin protoporphyrin IX, which is structurally related to pheophorbide a, were increased 10-fold. Transplantation with wild-type bone marrow cured the protoporphyria and reduced the phototoxin sensitivity of Bcrp1(-/-) mice. These results indicate that humans or animals with low or absent BCRP activity may be at increased risk for developing protoporphyria and diet-dependent phototoxicity and provide a striking illustration of the importance of drug transporters in protection from toxicity of normal food constituents.


Subject(s)
ATP-Binding Cassette Transporters/physiology , Chlorophyll/analogs & derivatives , Chlorophyll/toxicity , Dermatitis, Phototoxic/prevention & control , Drug Resistance/genetics , Neoplasm Proteins , Photosensitizing Agents/toxicity , Porphyria, Hepatoerythropoietic/prevention & control , Porphyrins/metabolism , Protoporphyrins/blood , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Administration, Oral , Animals , Bone Marrow Transplantation , Cell Line , Chlorophyll/administration & dosage , Chlorophyll/pharmacokinetics , Chromatography, High Pressure Liquid , Dermatitis, Phototoxic/etiology , Diet/adverse effects , Female , Fetus/metabolism , Fibroblasts/metabolism , Genetic Predisposition to Disease , Medicago sativa/adverse effects , Mice , Mice, Inbred Strains , Mice, Knockout , Molecular Structure , Photosensitizing Agents/administration & dosage , Photosensitizing Agents/pharmacokinetics , Porphyria, Hepatoerythropoietic/genetics , Porphyria, Hepatoerythropoietic/therapy , Porphyrins/pharmacokinetics , Pregnancy , Protoporphyrins/chemistry , Radiation Chimera , Topotecan/pharmacokinetics , Topotecan/toxicity
13.
Hum Mol Genet ; 11(3): 273-81, 2002 Feb 01.
Article in English | MEDLINE | ID: mdl-11823446

ABSTRACT

Fanconi anemia (FA) is a heterogeneous autosomal recessive chromosomal instability syndrome associated with diverse developmental abnormalities, progressive bone marrow failure and a predisposition to cancer. Spontaneous chromosomal breakage and hypersensitivity to DNA cross-linking agents characterize the cellular FA phenotype. The gene affected in FA complementation group G patients was initially identified as XRCC9, for its ability to partially correct the cellular phenotype of the Chinese hamster ovary (CHO) cell mutant UV40. By targeted disruption we generated Fancg/Xrcc9 null mice. Fancg knock-out (KO) mice were born at expected Mendelian frequencies and showed normal viability. In mice, functional loss of Fancg did not result in developmental abnormalities or a pronounced incidence of malignancies. During a 1 year follow-up, blood cell parameters of Fancg KO mice remained within normal values, revealing no signs of anemia. Male and female mice deficient in Fancg showed hypogonadism and impaired fertility, consistent with the phenotype of FA patients. Mouse embryonic fibroblasts (MEFs) from the KO animals exhibited the FA characteristic cellular response in showing enhanced spontaneous chromosomal instability and a hyper-responsiveness to the clastogenic and antiproliferative effects of the cross-linking agent mitomycin C (MMC). The sensitivity to UV, X-rays and methyl methanesulfonate, reported for the CHO mutant cell line UV40, was not observed in Fancg(-/-) MEFs. Despite a lack of hematopoietic failure in the KO mice, clonogenic survival of bone marrow cells in vitro was strongly reduced in the presence of MMC. The characteristics of the Fancg(-/-) mice closely resemble those reported for Fancc and Fanca null mice, supporting a tight interdependence of the corresponding gene products in a common pathway.


Subject(s)
DNA-Binding Proteins/genetics , Mitomycin/pharmacology , Animals , DNA/drug effects , DNA Damage , Drug Hypersensitivity , Fanconi Anemia/genetics , Fanconi Anemia Complementation Group G Protein , Female , Fibroblasts , Hematopoietic Stem Cells/drug effects , Infertility/genetics , Male , Mice , Mice, Knockout , Ovary/abnormalities , Testis/abnormalities
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