The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mi... more The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mice. Because the gp55 from the polycythemic strain (gp55-P), but not from the anemic strain (gp55-A), activates the erythropoietin receptor (EpoR) for proliferation of hematopoietic cell lines, the mechanism by which gp55-A initiates erythroleukemia has remained a mystery. We show here that gp55-A activates the EpoR in fetal liver cells. In contrast to previous studies using bone marrow cells from phenylhydrazine-treated, anemic mice, we find that both gp55-A and gp55-P induce erythroid differentiation from colony-forming unit-erythroid (CFU-E) progenitors in fetal liver cells. The effects on CFU-Es of both gp55-A and -P are mediated by the EpoR, because no colonies are seen upon expression of either gp55 in EpoR−/− fetal liver cells. However, only gp55-P induces erythroid bursts from burst-forming unit-erythroid progenitors and only gp55-P induces Epo independence in Epo-dependent cell l...
Proceedings of the National Academy of Sciences, 1999
Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characterist... more Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo −/− and EpoR −/− mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210 BCR-ABL and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR −/− mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of f...
Proceedings of the National Academy of Sciences, 2004
In the liver, insulin controls both lipid and glucose metabolism through its cell surface recepto... more In the liver, insulin controls both lipid and glucose metabolism through its cell surface receptor and intracellular mediators such as phosphatidylinositol 3-kinase and serine-threonine kinase AKT. The insulin signaling pathway is further modulated by protein tyrosine phosphatase or lipid phosphatase. Here, we investigated the function of phosphatase and tension homologue deleted on chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/AKT pathway, by targeted deletion of Pten in murine liver. Deletion of Pten in the liver resulted in increased fatty acid synthesis, accompanied by hepatomegaly and fatty liver phenotype. Interestingly, Pten liver-specific deletion causes enhanced liver insulin action with improved systemic glucose tolerance. Thus, deletion of Pten in the liver may provide a valuable model that permits the study of the metabolic actions of insulin signaling in the liver, and PTEN may be a promising target for therapeutic intervention for type...
In adipose tissue, insulin controls glucose and lipid metabolism through the intracellular mediat... more In adipose tissue, insulin controls glucose and lipid metabolism through the intracellular mediators phosphatidylinositol 3-kinase and serine-threonine kinase AKT. Phosphatase and a tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/AKT pathway, is hypothesized to inhibit the metabolic effects of insulin. Here we report the generation of mice lacking PTEN in adipose tissue. Loss of Pten results in improved systemic glucose tolerance and insulin sensitivity, associated with decreased fasting insulin levels, increased recruitment of the glucose transporter isoform 4 to the cell surface in adipose tissue, and decreased serum resistin levels. Mutant animals also exhibit increased insulin signaling and AMP kinase activity in the liver. Pten mutant mice are resistant to developing streptozotocin-induced diabetes. Adipose-specific Pten deletion, however, does not alter adiposity or plasma fatty acids. Our results demonstrate that in ...
PTEN is mutated at high frequency in many primary human cancers and several familial cancer predi... more PTEN is mutated at high frequency in many primary human cancers and several familial cancer predisposition disorders. Activation of AKT is a common event in tumors in which the PTEN gene has been inactivated. We previously showed that deletion of the murine Pten gene in embryonic stem (ES) cells led to increased phosphatidylinositol triphosphate (PIP 3 ) accumulation, enhanced entry into S phase, and better cell survival. Since PIP 3 controls multiple signaling molecules, it was not clear to what degree the observed phenotypes were due to deregulated AKT activity. In this study, we mutated Akt-1 in Pten −/− ES cells to directly assess the role of AKT-1 in PTEN-controlled cellular processes, such as cell proliferation, cell survival, and tumorigenesis in nude mice. We showed that AKT-1 is one of the major downstream effectors of PTEN in ES cells and that activation of AKT-1 is required for both the cell survival and cell proliferation phenotypes observed in Pten −/− ES cells. Deletio...
The ability to contribute to the germ line is the most important experimental feature of embryoni... more The ability to contribute to the germ line is the most important experimental feature of embryonic stem (ES) cells. Using ES cells, it is possible to introduce targeted mutations into any gene and to derive the corresponding mutant mice. A common problem with this technology is that the ES cells often lack or have only a low efficiency of germ line transmission. To address this issue, we examined the relationship between the growth rate and karyotype of ES cells, and their ability to contribute to the germ line. We found that chromosomal abnormalities occurred rather frequently in ES cells. Cells having an abnormal number of chromosomes, in particular trisomy 8, were found in three independently derived ES cell lines, and this abnormality conferred a selective growth advantage on these cells. Selection of abnormal cells led to depletion and eventual loss of normal ES cells during consecutive passages. In comparison with parental ES cells, ES cells with trisomy 8 contributed rarely to the germ line. This realization allowed us to select, based upon ES cell clone morphology, those clones with the highest probability of contributing to the germ line. This insight is of practical value for any given gene targeting experiment as it permits optimization of the rate of success without having to rely on more elaborate tests such as karyotyping individual clones prior to blastocyst injection.
To determine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in pancreas developmen... more To determine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in pancreas development, we generated a pancreas-specific knockout of Pten, a negative regulator of PI3-K signaling. Knockout mice display progressive replacement of the acinar pancreas with highly proliferative ductal structures that contain abundant mucins and express Pdx1 and Hes1, two markers of pancreatic progenitor cells. Moreover, a fraction of these mice develop ductal malignancy. We provide evidence that ductal metaplasia results from the expansion of centroacinar cells rather than transdifferentiation of acinar cells. These results indicate that Pten actively maintains the balance between different cell types in the adult pancreas and that misregulation of the PI3-K pathway in centroacinar cells may contribute to the initiation of pancreatic carcinoma in vivo.
Proceedings of the National Academy of Sciences, 2005
Previous studies have demonstrated that a small subpopulation of brain tumor cells share key char... more Previous studies have demonstrated that a small subpopulation of brain tumor cells share key characteristics with neural stem/progenitor cells in terms of phenotype and behavior. These findings suggest that brain tumors might contain “cancer stem cells” that are critical for tumor growth. However, the molecular pathways governing such stem cell-like behavior remain largely elusive. Our previous study suggests that the phosphatase and tensin homologue deleted on chromosome 10 ( PTEN ) tumor suppressor gene, one of the most frequently mutated genes in glioblastomas, restricts neural stem/progenitor cell proliferation in vivo . In the present study, we sought to determine the role of PTEN in long-term maintenance of stem cell-like properties, cell cycle entry and progression, and growth factor dependence and gene expression. Our results demonstrate an enhanced self-renewal capacity and G 0 -G 1 cell cycle entry and decreased growth factor dependency of Pten null neural/stem progenitor ...
Recent studies indicate that certain key molecules that are vital for various developmental proce... more Recent studies indicate that certain key molecules that are vital for various developmental processes, such as Wnt, Shh, and Notch, cause cancer when dysregulated. PTEN, a tumor suppressor that antagonizes the PI3 kinase pathway, is the newest one on the list. The biological function of PTEN is evolutionarily conserved from C. elegans to humans, and the PTEN-controlled signaling pathway regulates cellular processes crucial for normal development, including cell proliferation, soma growth, cell death, and cell migration. In this review, we will focus on the function of PTEN in murine development and its role in regulating stem cell self-renewal and proliferation. We will summarize the organomegaly phenotypes associated with Pten tissue-specific deletion and discuss how PTEN controls organ size, a fundamental aspect of development. Last, we will review the role of PTEN in hormone-dependent, adult-onset mammary and prostate gland development.
Proceedings of the National Academy of Sciences, 1999
To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutat... more To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten −/− ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G 1 /S transition was accompanied by down-regulation of p27 KIP1 , a major inhibitor for G 1 cyclin-dependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosage-dependent increases in phosphorylation and activation of Akt/protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten −/− cell survival. Our studies suggest that PTEN reg...
The potential role of stem cells in neoplasia is a subject of recent interest. Three markers of m... more The potential role of stem cells in neoplasia is a subject of recent interest. Three markers of melanocytic stem cells have been described recently. CD166 is expressed on the surface of mesenchymal stem cells and has been found on human melanoma cell lines. CD133 is expressed on the surface of dermal-derived stem cells that are capable of differentiating into neural cells. Nestin is an intermediate filament expressed in the cytoplasm of neuroepithelial stem cells. In this study, we evaluate the expression of these markers and possible differences among banal nevi, primary melanoma, and metastastic melanoma. Tissue microarrays containing normal tissue and 226 melanocytic lesions (71 banal nevi, 71 in situ and invasive melanomas, and 84 metastatic melanomas) were studied by immunohistochemistry using monoclonal antibodies CD166, CD133, and nestin. A significantly greater percentage of melanomas (combined primary and metastatic) contained cells that expressed CD166 (P ¼ 0.005), CD133 (P ¼ 0.003), and nestin (P ¼ 0.03) than banal nevi. Only nestin showed a statistical difference when comparing primary and metastatic melanoma (P ¼ 0.05). A stepwise increase in the proportion of lesions expressing all three markers was observed from banal nevi (2/19) to primary melanomas (8/17) to metastatic melanoma (19/28), P ¼ 0.0005. All cases of metastatic melanoma expressed at least one stem cell marker. The increased expression of CD166, CD133, and nestin in melanoma suggests that progression to malignant melanoma likely involves genetic pathways instrumental to stem cell biology and normal tissue development. Further studies and characterization of these pathways may also reveal new prognostic markers for a disease whose prognosis in advanced stages is dismal.
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase. PTEN inh... more Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase. PTEN inhibits the action of phosphatidylinositol-3-kinase and reduces the levels of phosphatidylinositol triphosphate, a crucial second messenger for cell proliferation and survival, as well as insulin signaling. In this study, we deleted Pten specifically in the insulin producing β cells during murine pancreatic development. Pten deletion leads to increased cell proliferation and decreased cell death, without significant alteration of β-cell differentiation. Consequently, the mutant pancreas generates more and larger islets, with a significant increase in total β-cell mass. PTEN loss also protects animals from developing streptozotocin-induced diabetes. Our data demonstrate that PTEN loss in β cells is not tumorigenic but beneficial. This suggests that modulating the PTEN-controlled signaling pathway is a potential approach for β-cell protection and regeneration therapies.
In the search for genes expressed in hematopoietic stem cells, we identified that the expression ... more In the search for genes expressed in hematopoietic stem cells, we identified that the expression of Gfi-1B (growth factor independence-1B) is highly restricted to hematopoietic stem cells, erythroblasts, and megakaryocytes. Gfi-1 and Gfi-1B are zinc finger proteins that share highly conserved SNAG and six zinc finger domains. Gfi-1 has been characterized as an oncogene involved in lymphoid malignancies in mice. In contrast, role of Gfi-1B in hematopoiesis has not been well characterized. In this study, we analyzed its function in human hematopoiesis. Enforced expression of Gfi-1B in human CD34 + hematopoietic progenitors induced a drastic expansion of erythroblasts in an erythropoietin-independent manner. Expression of Gfi-1B did not promote erythroid commitment, but enhanced proliferation of immature erythroblasts. Erythroblasts expanded by exogenous Gfi-1B, however, failed to differentiate beyond proerythroblast stage and showed massive apoptosis. These biological effects of Gfi-1B were mediated through its zinc finger domain, but not by the SNAG or non-zinc finger domain. Proliferation of erythroblasts was associated with sustained expression of GATA-2 but not of GATA-1, indicating a potential link between Gfi-1B and GATA family regulators. Importantly, the function of Gfi-1B to modulate transcription was dependent on promoter context. In addition, activation of transcription of an artificial promoter was mediated through its zinc finger domain. These findings establish Gfi-1B as a novel erythroid regulator and reveal its specific involvement in the regulation of erythroid cell growth through modulating erythroid-specific gene expression.
Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral... more Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin-sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogen...
Human germ cell tumors are often metastatic, presumably due to distal site tumor growth by cancer... more Human germ cell tumors are often metastatic, presumably due to distal site tumor growth by cancer stem cells. To determine whether cancer stem cells can be identified in a transplantation model of testicular germ cell tumor we transplanted murine embryonic germ cells (EGCs) into the testis of adult severe combined immunodeficient (SCID) mice. Transplantation resulted in a locally invasive solid tumor, with a cellular component that generated secondary tumors upon serial transplantation. The secondary tumors were invariably metastatic, a feature not observed in the primary tumors derived from EGCs. In order to characterize the differences between EGCs and the tumor-derived stem cells, we performed karyotype and microarray analysis. Our results show that generation of cancer stem cells is associated with the acquisition of non-clonal genomic rearrangements not found in the originating population. Furthermore, pretreatment of EGCs with a potent inhibitor of selfrenewal, retinoic acid, prevented tumor formation and the emergence of these genetically unstable cancer stem cells. Microarray analysis revealed that EGCs and first and second-generation cancer stem cells were highly similar, however approximately 1,000 differentiallyexpressed transcripts could be identified corresponding to alterations in oncogenes and genes associated with motility and development. Combined, the data suggest that the activation of oncogenic pathways in a cellular background of genetic instability coupled with an inherent ability to self-renew are involved in the acquisition of metastatic behavior in the cancer stem cell population of tumors derived from pluripotent cells.
10. In order to maximize the speed of image capture, we scan one focal section of the nuclear sph... more 10. In order to maximize the speed of image capture, we scan one focal section of the nuclear sphere every 1.5 s, and, if necessary, we adjust the objective between scans to keep the GFP-repressor spot in focus. The time required to capture lac op signal is ϳ15 ms, and the entire nucleus requires Ͻ150 ms. We analyse movies in which the plane of focus stays within a 1m midsection of the nucleus, or roughly half the nuclear depth. To ensure that cell cycle progression is not disturbed as a consequence of light damage, bud emergence and division of the scanned cell are followed by transmission microscopy.
Proceedings of the National Academy of Sciences, 2000
Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms... more Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk lo) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk ؊/؊ mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk lo phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk lo phenotype in vitro but not in vivo, whereas CD19 and the p85␣ form of phosphoinositide 3-kinase behaved as Btk lo enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85␣ haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C , Fyn, CD22, G␣q, or G␣11 had no detectable effect on the function of Btk lo B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.
Proceedings of the National Academy of Sciences, 1997
Type III collagen is a fibrillar forming collagen comprising three ␣1(III) chains and is expresse... more Type III collagen is a fibrillar forming collagen comprising three ␣1(III) chains and is expressed in early embryos and throughout embryogenesis. In the adult, type III collagen is a major component of the extracellular matrix in a variety of internal organs and skin. Mutations in the COL3A1 gene have been implicated as a cause of type IV Ehlers-Danlos syndrome, a disease leading to aortic rupture in early adult life. To directly study the role of Col3a1 in development and disease, we have inactivated the Col3a1 gene in embryonic stem cells by homologous recombination. The mutated allele was transmitted through the mouse germ line and homozygous mutant animals were derived from heterozygous intercrosses. About 10% of the homozygous mutant animals survived to adulthood but have a much shorter life span compared with wild-type mice. The major cause of death of mutant mice was rupture of the major blood vessels, similar to patients with type IV Ehlers-Danlos syndrome. Ultrastructural analysis of tissues from mutant mice revealed that type III collagen is essential for normal collagen I fibrillogenesis in the cardiovascular system and other organs. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mi... more The gp55 envelope proteins of the spleen focus-forming virus initiate erythroleukemia in adult mice. Because the gp55 from the polycythemic strain (gp55-P), but not from the anemic strain (gp55-A), activates the erythropoietin receptor (EpoR) for proliferation of hematopoietic cell lines, the mechanism by which gp55-A initiates erythroleukemia has remained a mystery. We show here that gp55-A activates the EpoR in fetal liver cells. In contrast to previous studies using bone marrow cells from phenylhydrazine-treated, anemic mice, we find that both gp55-A and gp55-P induce erythroid differentiation from colony-forming unit-erythroid (CFU-E) progenitors in fetal liver cells. The effects on CFU-Es of both gp55-A and -P are mediated by the EpoR, because no colonies are seen upon expression of either gp55 in EpoR−/− fetal liver cells. However, only gp55-P induces erythroid bursts from burst-forming unit-erythroid progenitors and only gp55-P induces Epo independence in Epo-dependent cell l...
Proceedings of the National Academy of Sciences, 1999
Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characterist... more Erythropoietin (Epo)-independent differentiation of erythroid progenitors is a major characteristic of myeloproliferative disorders, including chronic myeloid leukemia. Epo receptor (EpoR) signaling is crucial for normal erythroid development, as evidenced by the properties of Epo −/− and EpoR −/− mice, which contain a normal number of fetal liver erythroid progenitors but die in utero from a severe anemia attributable to the absence of red cell maturation. Here we show that two constitutively active cytoplasmic protein tyrosine kinases, P210 BCR-ABL and v-SRC, can functionally replace the EpoR and support full proliferation, differentiation, and maturation of fetal liver erythroid progenitors from EpoR −/− mice. These protein tyrosine kinases can also partially complement the myeloid growth factors IL-3, IL-6, and Steel factor, which are normally required in addition to Epo for erythroid development. Additionally, BCR-ABL mutants that lack residues necessary for transformation of f...
Proceedings of the National Academy of Sciences, 2004
In the liver, insulin controls both lipid and glucose metabolism through its cell surface recepto... more In the liver, insulin controls both lipid and glucose metabolism through its cell surface receptor and intracellular mediators such as phosphatidylinositol 3-kinase and serine-threonine kinase AKT. The insulin signaling pathway is further modulated by protein tyrosine phosphatase or lipid phosphatase. Here, we investigated the function of phosphatase and tension homologue deleted on chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/AKT pathway, by targeted deletion of Pten in murine liver. Deletion of Pten in the liver resulted in increased fatty acid synthesis, accompanied by hepatomegaly and fatty liver phenotype. Interestingly, Pten liver-specific deletion causes enhanced liver insulin action with improved systemic glucose tolerance. Thus, deletion of Pten in the liver may provide a valuable model that permits the study of the metabolic actions of insulin signaling in the liver, and PTEN may be a promising target for therapeutic intervention for type...
In adipose tissue, insulin controls glucose and lipid metabolism through the intracellular mediat... more In adipose tissue, insulin controls glucose and lipid metabolism through the intracellular mediators phosphatidylinositol 3-kinase and serine-threonine kinase AKT. Phosphatase and a tensin homolog deleted from chromosome 10 (PTEN), a negative regulator of the phosphatidylinositol 3-kinase/AKT pathway, is hypothesized to inhibit the metabolic effects of insulin. Here we report the generation of mice lacking PTEN in adipose tissue. Loss of Pten results in improved systemic glucose tolerance and insulin sensitivity, associated with decreased fasting insulin levels, increased recruitment of the glucose transporter isoform 4 to the cell surface in adipose tissue, and decreased serum resistin levels. Mutant animals also exhibit increased insulin signaling and AMP kinase activity in the liver. Pten mutant mice are resistant to developing streptozotocin-induced diabetes. Adipose-specific Pten deletion, however, does not alter adiposity or plasma fatty acids. Our results demonstrate that in ...
PTEN is mutated at high frequency in many primary human cancers and several familial cancer predi... more PTEN is mutated at high frequency in many primary human cancers and several familial cancer predisposition disorders. Activation of AKT is a common event in tumors in which the PTEN gene has been inactivated. We previously showed that deletion of the murine Pten gene in embryonic stem (ES) cells led to increased phosphatidylinositol triphosphate (PIP 3 ) accumulation, enhanced entry into S phase, and better cell survival. Since PIP 3 controls multiple signaling molecules, it was not clear to what degree the observed phenotypes were due to deregulated AKT activity. In this study, we mutated Akt-1 in Pten −/− ES cells to directly assess the role of AKT-1 in PTEN-controlled cellular processes, such as cell proliferation, cell survival, and tumorigenesis in nude mice. We showed that AKT-1 is one of the major downstream effectors of PTEN in ES cells and that activation of AKT-1 is required for both the cell survival and cell proliferation phenotypes observed in Pten −/− ES cells. Deletio...
The ability to contribute to the germ line is the most important experimental feature of embryoni... more The ability to contribute to the germ line is the most important experimental feature of embryonic stem (ES) cells. Using ES cells, it is possible to introduce targeted mutations into any gene and to derive the corresponding mutant mice. A common problem with this technology is that the ES cells often lack or have only a low efficiency of germ line transmission. To address this issue, we examined the relationship between the growth rate and karyotype of ES cells, and their ability to contribute to the germ line. We found that chromosomal abnormalities occurred rather frequently in ES cells. Cells having an abnormal number of chromosomes, in particular trisomy 8, were found in three independently derived ES cell lines, and this abnormality conferred a selective growth advantage on these cells. Selection of abnormal cells led to depletion and eventual loss of normal ES cells during consecutive passages. In comparison with parental ES cells, ES cells with trisomy 8 contributed rarely to the germ line. This realization allowed us to select, based upon ES cell clone morphology, those clones with the highest probability of contributing to the germ line. This insight is of practical value for any given gene targeting experiment as it permits optimization of the rate of success without having to rely on more elaborate tests such as karyotyping individual clones prior to blastocyst injection.
To determine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in pancreas developmen... more To determine the role of the phosphatidylinositol 3-kinase (PI3-K) pathway in pancreas development, we generated a pancreas-specific knockout of Pten, a negative regulator of PI3-K signaling. Knockout mice display progressive replacement of the acinar pancreas with highly proliferative ductal structures that contain abundant mucins and express Pdx1 and Hes1, two markers of pancreatic progenitor cells. Moreover, a fraction of these mice develop ductal malignancy. We provide evidence that ductal metaplasia results from the expansion of centroacinar cells rather than transdifferentiation of acinar cells. These results indicate that Pten actively maintains the balance between different cell types in the adult pancreas and that misregulation of the PI3-K pathway in centroacinar cells may contribute to the initiation of pancreatic carcinoma in vivo.
Proceedings of the National Academy of Sciences, 2005
Previous studies have demonstrated that a small subpopulation of brain tumor cells share key char... more Previous studies have demonstrated that a small subpopulation of brain tumor cells share key characteristics with neural stem/progenitor cells in terms of phenotype and behavior. These findings suggest that brain tumors might contain “cancer stem cells” that are critical for tumor growth. However, the molecular pathways governing such stem cell-like behavior remain largely elusive. Our previous study suggests that the phosphatase and tensin homologue deleted on chromosome 10 ( PTEN ) tumor suppressor gene, one of the most frequently mutated genes in glioblastomas, restricts neural stem/progenitor cell proliferation in vivo . In the present study, we sought to determine the role of PTEN in long-term maintenance of stem cell-like properties, cell cycle entry and progression, and growth factor dependence and gene expression. Our results demonstrate an enhanced self-renewal capacity and G 0 -G 1 cell cycle entry and decreased growth factor dependency of Pten null neural/stem progenitor ...
Recent studies indicate that certain key molecules that are vital for various developmental proce... more Recent studies indicate that certain key molecules that are vital for various developmental processes, such as Wnt, Shh, and Notch, cause cancer when dysregulated. PTEN, a tumor suppressor that antagonizes the PI3 kinase pathway, is the newest one on the list. The biological function of PTEN is evolutionarily conserved from C. elegans to humans, and the PTEN-controlled signaling pathway regulates cellular processes crucial for normal development, including cell proliferation, soma growth, cell death, and cell migration. In this review, we will focus on the function of PTEN in murine development and its role in regulating stem cell self-renewal and proliferation. We will summarize the organomegaly phenotypes associated with Pten tissue-specific deletion and discuss how PTEN controls organ size, a fundamental aspect of development. Last, we will review the role of PTEN in hormone-dependent, adult-onset mammary and prostate gland development.
Proceedings of the National Academy of Sciences, 1999
To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutat... more To investigate the molecular basis of PTEN-mediated tumor suppression, we introduced a null mutation into the mouse Pten gene by homologous recombination in embryonic stem (ES) cells. Pten −/− ES cells exhibited an increased growth rate and proliferated even in the absence of serum. ES cells lacking PTEN function also displayed advanced entry into S phase. This accelerated G 1 /S transition was accompanied by down-regulation of p27 KIP1 , a major inhibitor for G 1 cyclin-dependent kinases. Inactivation of PTEN in ES cells and in embryonic fibroblasts resulted in elevated levels of phosphatidylinositol 3,4,5,-trisphosphate, a product of phosphatidylinositol 3 kinase. Consequently, PTEN deficiency led to dosage-dependent increases in phosphorylation and activation of Akt/protein kinase B, a well-characterized target of the phosphatidylinositol 3 kinase signaling pathway. Akt activation increased Bad phosphorylation and promoted Pten −/− cell survival. Our studies suggest that PTEN reg...
The potential role of stem cells in neoplasia is a subject of recent interest. Three markers of m... more The potential role of stem cells in neoplasia is a subject of recent interest. Three markers of melanocytic stem cells have been described recently. CD166 is expressed on the surface of mesenchymal stem cells and has been found on human melanoma cell lines. CD133 is expressed on the surface of dermal-derived stem cells that are capable of differentiating into neural cells. Nestin is an intermediate filament expressed in the cytoplasm of neuroepithelial stem cells. In this study, we evaluate the expression of these markers and possible differences among banal nevi, primary melanoma, and metastastic melanoma. Tissue microarrays containing normal tissue and 226 melanocytic lesions (71 banal nevi, 71 in situ and invasive melanomas, and 84 metastatic melanomas) were studied by immunohistochemistry using monoclonal antibodies CD166, CD133, and nestin. A significantly greater percentage of melanomas (combined primary and metastatic) contained cells that expressed CD166 (P ¼ 0.005), CD133 (P ¼ 0.003), and nestin (P ¼ 0.03) than banal nevi. Only nestin showed a statistical difference when comparing primary and metastatic melanoma (P ¼ 0.05). A stepwise increase in the proportion of lesions expressing all three markers was observed from banal nevi (2/19) to primary melanomas (8/17) to metastatic melanoma (19/28), P ¼ 0.0005. All cases of metastatic melanoma expressed at least one stem cell marker. The increased expression of CD166, CD133, and nestin in melanoma suggests that progression to malignant melanoma likely involves genetic pathways instrumental to stem cell biology and normal tissue development. Further studies and characterization of these pathways may also reveal new prognostic markers for a disease whose prognosis in advanced stages is dismal.
Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase. PTEN inh... more Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a lipid phosphatase. PTEN inhibits the action of phosphatidylinositol-3-kinase and reduces the levels of phosphatidylinositol triphosphate, a crucial second messenger for cell proliferation and survival, as well as insulin signaling. In this study, we deleted Pten specifically in the insulin producing β cells during murine pancreatic development. Pten deletion leads to increased cell proliferation and decreased cell death, without significant alteration of β-cell differentiation. Consequently, the mutant pancreas generates more and larger islets, with a significant increase in total β-cell mass. PTEN loss also protects animals from developing streptozotocin-induced diabetes. Our data demonstrate that PTEN loss in β cells is not tumorigenic but beneficial. This suggests that modulating the PTEN-controlled signaling pathway is a potential approach for β-cell protection and regeneration therapies.
In the search for genes expressed in hematopoietic stem cells, we identified that the expression ... more In the search for genes expressed in hematopoietic stem cells, we identified that the expression of Gfi-1B (growth factor independence-1B) is highly restricted to hematopoietic stem cells, erythroblasts, and megakaryocytes. Gfi-1 and Gfi-1B are zinc finger proteins that share highly conserved SNAG and six zinc finger domains. Gfi-1 has been characterized as an oncogene involved in lymphoid malignancies in mice. In contrast, role of Gfi-1B in hematopoiesis has not been well characterized. In this study, we analyzed its function in human hematopoiesis. Enforced expression of Gfi-1B in human CD34 + hematopoietic progenitors induced a drastic expansion of erythroblasts in an erythropoietin-independent manner. Expression of Gfi-1B did not promote erythroid commitment, but enhanced proliferation of immature erythroblasts. Erythroblasts expanded by exogenous Gfi-1B, however, failed to differentiate beyond proerythroblast stage and showed massive apoptosis. These biological effects of Gfi-1B were mediated through its zinc finger domain, but not by the SNAG or non-zinc finger domain. Proliferation of erythroblasts was associated with sustained expression of GATA-2 but not of GATA-1, indicating a potential link between Gfi-1B and GATA family regulators. Importantly, the function of Gfi-1B to modulate transcription was dependent on promoter context. In addition, activation of transcription of an artificial promoter was mediated through its zinc finger domain. These findings establish Gfi-1B as a novel erythroid regulator and reveal its specific involvement in the regulation of erythroid cell growth through modulating erythroid-specific gene expression.
Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral... more Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin-sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogen...
Human germ cell tumors are often metastatic, presumably due to distal site tumor growth by cancer... more Human germ cell tumors are often metastatic, presumably due to distal site tumor growth by cancer stem cells. To determine whether cancer stem cells can be identified in a transplantation model of testicular germ cell tumor we transplanted murine embryonic germ cells (EGCs) into the testis of adult severe combined immunodeficient (SCID) mice. Transplantation resulted in a locally invasive solid tumor, with a cellular component that generated secondary tumors upon serial transplantation. The secondary tumors were invariably metastatic, a feature not observed in the primary tumors derived from EGCs. In order to characterize the differences between EGCs and the tumor-derived stem cells, we performed karyotype and microarray analysis. Our results show that generation of cancer stem cells is associated with the acquisition of non-clonal genomic rearrangements not found in the originating population. Furthermore, pretreatment of EGCs with a potent inhibitor of selfrenewal, retinoic acid, prevented tumor formation and the emergence of these genetically unstable cancer stem cells. Microarray analysis revealed that EGCs and first and second-generation cancer stem cells were highly similar, however approximately 1,000 differentiallyexpressed transcripts could be identified corresponding to alterations in oncogenes and genes associated with motility and development. Combined, the data suggest that the activation of oncogenic pathways in a cellular background of genetic instability coupled with an inherent ability to self-renew are involved in the acquisition of metastatic behavior in the cancer stem cell population of tumors derived from pluripotent cells.
10. In order to maximize the speed of image capture, we scan one focal section of the nuclear sph... more 10. In order to maximize the speed of image capture, we scan one focal section of the nuclear sphere every 1.5 s, and, if necessary, we adjust the objective between scans to keep the GFP-repressor spot in focus. The time required to capture lac op signal is ϳ15 ms, and the entire nucleus requires Ͻ150 ms. We analyse movies in which the plane of focus stays within a 1m midsection of the nucleus, or roughly half the nuclear depth. To ensure that cell cycle progression is not disturbed as a consequence of light damage, bud emergence and division of the scanned cell are followed by transmission microscopy.
Proceedings of the National Academy of Sciences, 2000
Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms... more Modifier screens have been powerful genetic tools to define signaling pathways in lower organisms. The identification of modifier loci in mice has begun to allow a similar dissection of mammalian signaling pathways. Transgenic mice (Btk lo) expressing 25% of endogenous levels of Bruton's tyrosine kinase (Btk) have B cell functional responses between those of wild-type and Btk ؊/؊ mice. We asked whether reduced dosage or complete deficiency of genes previously implicated as Btk regulators would modify the Btk lo phenotype. We used two independent assays of Btk-dependent B cell function. Proliferative response to B cell antigen receptor cross-linking in vitro was chosen as an example of a relatively simple, well-defined signaling system. In vivo response to type II T-independent antigens (TI-II) measures complex interactions among multiple cell types over time and may identify additional Btk pathways. All modifiers identified differentially affected these two assays, indicating that Btk mediates these processes via distinct mechanisms. Loss of Lyn, PTEN (phosphatase and tensin homolog), or SH2-containing inositol phosphatase suppressed the Btk lo phenotype in vitro but not in vivo, whereas CD19 and the p85␣ form of phosphoinositide 3-kinase behaved as Btk lo enhancers in vivo but not in vitro. Effects of Lyn, PTEN, or p85␣ haploinsufficiency were observed. Haploinsufficiency or complete deficiency of protein kinase C , Fyn, CD22, G␣q, or G␣11 had no detectable effect on the function of Btk lo B cells. A transgenic system creating a reduction in dosage of Btk can therefore be used to identify modifier loci that affect B cell responses and quantitatively rank their contribution to Btk-mediated processes.
Proceedings of the National Academy of Sciences, 1997
Type III collagen is a fibrillar forming collagen comprising three ␣1(III) chains and is expresse... more Type III collagen is a fibrillar forming collagen comprising three ␣1(III) chains and is expressed in early embryos and throughout embryogenesis. In the adult, type III collagen is a major component of the extracellular matrix in a variety of internal organs and skin. Mutations in the COL3A1 gene have been implicated as a cause of type IV Ehlers-Danlos syndrome, a disease leading to aortic rupture in early adult life. To directly study the role of Col3a1 in development and disease, we have inactivated the Col3a1 gene in embryonic stem cells by homologous recombination. The mutated allele was transmitted through the mouse germ line and homozygous mutant animals were derived from heterozygous intercrosses. About 10% of the homozygous mutant animals survived to adulthood but have a much shorter life span compared with wild-type mice. The major cause of death of mutant mice was rupture of the major blood vessels, similar to patients with type IV Ehlers-Danlos syndrome. Ultrastructural analysis of tissues from mutant mice revealed that type III collagen is essential for normal collagen I fibrillogenesis in the cardiovascular system and other organs. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
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