ABSTRACT
Flotillin microdomains, specialized lipid raft domains in cell membranes, serve as physical platforms for many different molecules important in crucial intracellular signaling pathways. Flotillin-2 (Flot2), together with flotillin-1, is a marker for lipid raft microdomains distinct from caveolar lipid rafts, and has been implicated in the progression of cancer and metastasis formation. Based largely on studies in xenograft models, flotillin-2 has been implicated in the progression of multiple types of human tumors, including breast cancer. In our studies, we identified flotillin-2 as highly amplified in a high-throughput comparative genomic hybridization screen of human breast cancer cell lines and breast tumor samples. Short hairpin RNA-mediated reduction of flotillin-2 protein levels significantly reduced the tumorigenicity and metastatic capability of a human breast cancer cell line in vivo. We generated mice deficient for flotillin-2 and also found a reduction of flotillin-1 protein levels and complete absence of flotillin-specific membrane microdomains in these mice. To examine the role of Flot2 in mammary tumorigenesis and lung metastasis, we used an in vivo molecular genetics approach, crossing a well-characterized transgenic mouse model of breast cancer, the MMTV-PyMT (mouse mammary tumor virus-polyoma middle T antigen) mouse, with gene-targeted Flot2(-/-) mice. Flotillin-2 deficiency lead to a striking reduction in the number of lung metastasis observed, but had no influence on primary tumor formation in this model. Our results indicate, using a novel in vivo animal model approach, that Flot2 is an important regulator of mammary tumor-derived lung metastasis.
Subject(s)
Lung Neoplasms/genetics , Lung Neoplasms/secondary , Mammary Neoplasms, Experimental/pathology , Membrane Proteins/deficiency , Membrane Proteins/genetics , Animals , Cell Line, Tumor , Cell Transformation, Neoplastic , Comparative Genomic Hybridization , Disease Models, Animal , Female , Gene Silencing , Humans , MiceABSTRACT
Proteins containing a caveolin-binding domain (CBD), such as the Rho-GTPases, can interact with caveolin-1 (Cav1) through its caveolin scaffold domain. Rho-GTPases are important regulators of p130(Cas), which is crucial for both normal cell migration and Src kinase-mediated metastasis of cancer cells. However, although Rho-GTPases (particularly RhoC) and Cav1 have been linked to cancer progression and metastasis, the underlying molecular mechanisms are largely unknown. To investigate the function of Cav1-Rho-GTPase interaction in metastasis, we disrupted Cav1-Rho-GTPase binding in melanoma and mammary epithelial tumor cells by overexpressing CBD, and examined the loss-of-function of RhoC in metastatic cancer cells. Cancer cells overexpressing CBD or lacking RhoC had reduced p130(Cas) phosphorylation and Rac1 activation, resulting in an inhibition of migration and invasion in vitro. The activity of Src and the activation of its downstream targets FAK, Pyk2, Ras and extracellular signal-regulated kinase (Erk)1/2 were also impaired. A reduction in α5-integrin expression, which is required for binding to fibronectin and thus cell migration and survival, was observed in CBD-expressing cells and cells lacking RhoC. As a result of these defects, CBD-expressing melanoma cells had a reduced ability to metastasize in recipient mice, and impaired extravasation and survival in secondary sites in chicken embryos. Our data indicate that interaction between Cav1 and Rho-GTPases (most likely RhoC but not RhoA) promotes metastasis by stimulating α5-integrin expression and regulating the Src-dependent activation of p130(Cas)/Rac1, FAK/Pyk2 and Ras/Erk1/2 signaling cascades.
Subject(s)
Caveolin 1/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Integrin alpha5/metabolism , ras Proteins/metabolism , rho GTP-Binding Proteins/metabolism , src-Family Kinases/metabolism , Amino Acid Sequence , Animals , Caveolin 1/genetics , Cell Line, Tumor , Cell Movement , Chick Embryo , Crk-Associated Substrate Protein/genetics , Crk-Associated Substrate Protein/metabolism , Enzyme Activation , Extracellular Signal-Regulated MAP Kinases/genetics , Immunoblotting , Integrin alpha5/genetics , Mice , Mice, Inbred C57BL , Mice, SCID , Molecular Sequence Data , Neoplasm Metastasis , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Phosphorylation , Protein Binding , RNA Interference , Sequence Homology, Amino Acid , ras Proteins/genetics , rho GTP-Binding Proteins/genetics , rhoC GTP-Binding Protein , src-Family Kinases/geneticsSubject(s)
Carrier Proteins/genetics , Membrane Transport Proteins , Mutation, Missense , Osteochondrodysplasias/genetics , X Chromosome , Adolescent , Adult , Age of Onset , Aged , Amino Acid Sequence , Carrier Proteins/chemistry , Child , Female , Genetic Linkage , Heterozygote , Humans , Male , Middle Aged , Molecular Sequence Data , Osteochondrodysplasias/diagnosis , Pedigree , Protein Structure, Secondary , Transcription FactorsABSTRACT
We generated purine nucleoside phosphorylase (PNP)-deficient mice to gain insight into the mechanism of immune deficiency disease associated with PNP deficiency in humans. Similar to the human disease, PNP deficiency in mice causes an immunodeficiency that affects T lymphocytes more severely than B lymphocytes. PNP knockout mice exhibit impaired thymocyte differentiation, reduced mitogenic and allogeneic responses, and decreased numbers of maturing thymocytes and peripheral T cells. T lymphocytes of PNP-deficient mice exhibit increased apoptosis in vivo and higher sensitivity to gamma irradiation in vitro. We propose that the immune deficiency in PNP deficiency is a result of inhibition of mitochondrial DNA repair due to the accumulation of dGTP in the mitochondria. The end result is increased sensitivity of T cells to spontaneous mitochondrial DNA damage, leading to T cell depletion by apoptosis.
Subject(s)
Deoxyguanine Nucleotides/metabolism , Mitochondria/metabolism , Purine-Nucleoside Phosphorylase/deficiency , Purine-Nucleoside Phosphorylase/genetics , Severe Combined Immunodeficiency/etiology , T-Lymphocytes/metabolism , Animals , Apoptosis , CD4-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/metabolism , Cell Differentiation , Cytotoxicity, Immunologic , Mice , Mice, Knockout , T-Lymphocyte Subsets/metabolism , Thymus Gland/cytologySubject(s)
Inosine/analogs & derivatives , Purine-Nucleoside Phosphorylase/physiology , Animals , Antigens, CD19 , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Biomarkers , CD4-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/cytology , Deoxyguanine Nucleotides/metabolism , Deoxyguanosine/urine , Guanosine/urine , Guanosine Triphosphate/metabolism , Inosine/urine , Mice , Mice, Knockout , Purine-Nucleoside Phosphorylase/genetics , Purine-Nucleoside Phosphorylase/immunologyABSTRACT
Members of the Jak family play a critical role in signal transduction mediated by cytokine and hormone receptors. In this study, we report the cloning and characterization of human Jak2. The predicted amino acid sequence shows 91% homology to the described murine Jak2, but with a significant difference in the extreme C-terminal sequence. Using the human cDNA as a probe, we localized the gene for human Jak2 to chromosome 9p23-24. Human Jak2 mRNA is highly expressed in the spleen, lymph nodes, and peripheral blood lymphocytes (PBLs). A polyclonal antibody raised against the unique C-terminus of human Jak2 was used to characterize Jak2 protein. Levels of Jak2 protein expression increased significantly in mitogen- and anti-IgM-stimulated B cells and to a lesser degree in activated T cells. In addition, high levels of Jak2 protein were detected in pre-B leukemia cells.
Subject(s)
Cloning, Molecular , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Chromosomes, Human, Pair 9 , DNA Probes , Gene Expression , Hematopoietic Stem Cells/metabolism , Humans , Janus Kinase 2 , Lymphoid Tissue/metabolism , Mice , Molecular Sequence Data , Protein-Tyrosine Kinases/chemistry , RNA, Messenger/analysis , Sequence Homology , Signal Transduction , Tissue DistributionABSTRACT
Acute lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Despite the progress achieved in its treatment, 20% of cases relapse and no longer respond to chemotherapy. The most common phenotype of ALL cells share surface antigens with very early precursors of B cells and are therefore believed to originate from this lineage. Characterization of the growth requirement of ALL cells indicated that they were dependent on various cytokines, suggesting paracrine and/or autocrine growth regulation. Because many cytokines induce tyrosine phosphorylation in lymphoid progenitor cells, and constitutive tyrosine phosphorylation is commonly observed in B-lineage leukaemias, attempts have been made to develop protein tyrosine kinase (PTK) blockers of leukaemia cell growth. Here we show that leukaemic cells from patients in relapse have constitutively activated Jak-2 PTK. Inhibition of Jak-2 activity by a specific tyrosine kinase blocker, AG-490, selectively blocks leukaemic cell growth in vitro and in vivo by inducing programmed cell death, with no deleterious effect on normal haematopoiesis.
Subject(s)
Antineoplastic Agents/pharmacology , Enzyme Inhibitors/pharmacology , Nitriles/pharmacology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins , Tyrphostins , Animals , Antineoplastic Agents/chemistry , B-Lymphocytes/drug effects , Cell Division/drug effects , Humans , Janus Kinase 2 , Mice , Mice, SCID , Neoplasm Transplantation , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Structure-Activity Relationship , Tumor Cells, CulturedABSTRACT
We have previously described a type of selective T cell deficiency (STD) characterized by persistent infections reminiscent of severe combined immunodeficiency. We show here that STD patients carry a mutation of zap-70, resulting in loss of the activity of this kinase. The thymi of zap-70-/- patients show the presence of CD4+CD8+ cells in the cortex; however, only CD4, not CD8, single-positive cells are present in the medulla. Peripheral CD4+ T cells from the zap-70-/- patients exhibit markedly reduced tyrosine phosphorylation, fail to produce interleukin-2, and do not proliferate in response to T cell receptor stimulation by mitogens or antigens. Thus, Zap-70 kinase appears to be indispensable for the development of CD8 single-positive T cells as well as for signal transduction and function of single-positive CD4 T cells.
Subject(s)
CD8 Antigens/metabolism , Immunologic Deficiency Syndromes/genetics , Protein-Tyrosine Kinases/metabolism , Protein-Tyrosine Kinases/physiology , Receptors, Antigen, T-Cell, alpha-beta/physiology , T-Lymphocyte Subsets/cytology , Thymus Gland/cytology , Amino Acid Sequence , Cell Differentiation , Female , Heterozygote , Humans , Immunologic Deficiency Syndromes/enzymology , Male , Molecular Sequence Data , Pedigree , Signal Transduction , T-Lymphocyte Subsets/enzymology , ZAP-70 Protein-Tyrosine KinaseABSTRACT
Tay-Sachs disease is an autosomal recessive genetic disorder resulting from mutation of the HEXA gene encoding the alpha-subunit of the lysosomal enzyme, beta-N-acetylhexosaminidase A (ref. 1). A relatively high frequency of carriers (1/27) of a lethal, infantile form of the disease is found in the Ashkenazi Jewish population, but it is not yet evident whether this has resulted from a founder effect and random genetic drift or from a selective advantage of heterozygotes. We have identified a single-base mutation in a cloned fragment of the HEXA gene from an Ashkenazi Jewish patient. This change, the substitution of a C for G in the first nucleotide of intron 12 is expected to result in defective splicing of the messenger RNA. A test for the mutant allele based on amplification of DNA by the 'polymerase chain rection and cleavage of a DdeI restriction site generated by the mutation revealed that this case and two other cases of the Ashkenazi, infantile form of Tay-Sachs disease are heterozygous for two different mutations. The occurrence of multiple mutant alleles warrants further examination of the selective advantage hypothesis.
Subject(s)
DNA/genetics , RNA Splicing , RNA, Messenger/genetics , Tay-Sachs Disease/genetics , Base Sequence , Cell Line , Cloning, Molecular , Female , Gene Amplification , Humans , Male , Mutation , PedigreeABSTRACT
Three classes of mutants resistant to the drug 6(p-hydroxyphenylazo)-uracil have been isolated from mutagenized cultures of CHO cells. One class of these mutants designated HPURA exhibits a unique form of cross-resistance to bromodeoxyuridine in that it is resistant to this drug only in the presence of thymidine. The molecular basis of the BrdU resistance is unknown but does not appear to involve the known targets of the drug. An interesting feature of these mutants is that they give rise, at a high frequency, to a subpopulation of cells which are much more resistant to BrdU.
Subject(s)
Bromodeoxyuridine/pharmacology , Hydroxyphenylazouracil/pharmacology , Mutation , Uracil/analogs & derivatives , Animals , Biological Transport , Cell Line , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Drug Resistance , Female , Hydroxyphenylazouracil/metabolism , Kinetics , Ovary , Phenotype , Thymidine/metabolismABSTRACT
Two classes of CHO mutants resistant to the drug 6(p-hydroxyphenylazo)-uracil have been characterized. Both classes exhibited a nutritional requirement that could be satisfied by deoxypyrimidines and uridine but not other ribopyrimidines. A biochemical investigation of these mutants revealed a structural defect in ribonucleotide reductase resulting in a two- to fourfold increase in the Km for UDP and CDP. As a consequence of this lesion, the cells had imbalanced deoxypyrimidine pools and showed an increase in the rate of spontaneous mutation to 6-thioguanine resistance but not emetine resistance.
Subject(s)
Deoxyribonucleosides/metabolism , Hydroxyphenylazouracil/pharmacology , Mutation , Uracil/analogs & derivatives , Animals , Cell Cycle/drug effects , Cell Line , Cricetinae , Cricetulus , Drug Resistance , Female , Kinetics , Ovary , Ribonucleotides/metabolismABSTRACT
Data presented here on the quantitative 3H-thymidine incorporation into DNA, after PHA mitogenic stimulation, show that 21-trisomic lymphocytes are low-responders to PHA compared with the normal-diploid ones. Their responsiveness seems to decrease with the donor's age. Auto-radiographic studies clearly demonstrate that the fraction of labeled cells at the 72nd h of incubation is significantly smaller in the 21-trisomic lymphocyte population. The comparison of labeling indexes at different times of incubation (24, 48, 72 h) also indicate, in the same population, a slower increment of the portion of DNA-synthesizing cells. Discussing these data in the light of other's observations and recent progress in the knowledge of factors and mechanisms involved in the lymphocyte response to lectin mitogenic stimulus, it is suggested that differential distribution of T- and B- and/or T-cell subpopulations and a retarded cell induction time to proliferate may be two important factors negatively influencing the responsiveness of 21-trisomic lymphocyte population.