Poster Session-Drug targets which provides a Dolichol Phosphate (DolP) substitute on regulation o... more Poster Session-Drug targets which provides a Dolichol Phosphate (DolP) substitute on regulation of Pgp expression in Doxorubicin resistant MCF-7 breast cancer cells. Methods: Breast cancer cell lines, MCF-7 and MCF-7/ADR were used. Pol concentration in the culture medium made up 10 −2-10 −6. Immunohistochemical and Western blotting methods were used to detect the changes in the expression levels of MDR1 and DPAGT1 expression. Intermediates of DPC fractions were analysed by HPLC method. Results: Overexpression of DPAGT1 was detected MCF-7/ADR cells, but not in MCF-7 cells. It is confirmed that plasmatic membranes of MCF-7 cells contain 5.6-6.4% of Pgp (the total protein amount) as a resistance marker. Resistant MCF-7/ADR cells differ from sensitive ones MCF-7 in Pgp content by 10−12 times. The study showed 8.5-fold DolP decrease in MCF-7/ADR cells. The investigations demonstrate that the situation can be changed by treatment with DolP and PP. The DolP concentration in MCF-7/ADR cells was returned to the normal level. It is established that DolP in the concentration 10 −6 M aid 7−9-fold reducing Pgp in membranes of MCF-7/ADR cells. The MCF-7/ADR cells cultivation in medium with polyprenol proceeded to give lowered Pgp content in membranes no over 0.4−0.6%, which amount was consistent with the level of Pgp in MCF-7 cells. Overexpression of DPAGT1 was detected in MCF-7 and in MCF-7/ADR cells. It is established that Pol in the concentration 10 −4 M aid 7−9-fold could overcome DPAGT1 overexpression which leads to regulation of Pgp N-glycosylation. Pol in concentration 10 −2 −10 −3 M induced apoptosis in MCF-7/ADR cells within 3−4 hours. Conclusions: These results indicate that noncontrollable accumulation of Pgp, after MDR1 expression in MCF-7/ADR cells can be overcome using stimulation with dolichyl phosphate substitution. DPAGT1 overexpression in MCF-7/ADR can be overcome with Pol, which provides a DolP substitute for DPAGT1 normal expression.
We have used gene targeted mutational approaches to assess the role of the T cell receptor alpha ... more We have used gene targeted mutational approaches to assess the role of the T cell receptor alpha (TCR alpha) enhancer (E alpha) in the control of TCR alpha and TCR delta gene rearrangement and expression. We show that E alpha functions in cis to promote V alpha to J alpha rearrangement across the entire J alpha locus, a distance of greater than 70 kb. We also show that E alpha is required for normal alphabeta T cell development; in this lineage, E alpha is required for germline J alpha expression, for normal expression levels of rearranged V alpha J alpha genes, and for expression of a diverse V alpha repertoire. In gamma delta T cells, E alpha is not required for VdeltaDJdelta rearrangement, but, surprisingly, is required for normal expression levels of mature VdeltaDJdelta transcripts and for expression of germline J alpha transcripts. Our findings imply that E alpha function is not limited to the TCR alpha components of the TCRalpha/delta locus or to the alpha beta lineage; rathe...
Summary The 40-kb region downstream of the most 3 9 immunoglobulin (Ig) heavy chain constant re- ... more Summary The 40-kb region downstream of the most 3 9 immunoglobulin (Ig) heavy chain constant re- gion gene (C a ) contains a series of transcriptional enhancers speculated to play a role in Ig heavy chain class switch recombination (CSR). To elucidate the function of this putative CSR regulatory region, we generated mice with germline mutations in which one or the other of the two most 5 9 enhancers in this cluster (respectively referred to as HS3a and HS1,2) were re- placed either with a pgk- neo r cassette (referred to as HS3aN and HS1,2N mutations) or with a loxP sequence (referred to as HS3a D and HS1,2 D , respectively). B cells homozygous for the HS3aN or HS1,2N mutations had severe defects in CSR to several isotypes. The phenotypic similarity of the two insertion mutations, both of which were cis -acting, suggested that inhibi- tion might result from pgk- neo r cassette gene insertion rather than enhancer deletion. Accord- ingly, CSR returned to normal in B cells homozygous f...
Proceedings of the National Academy of Sciences, 1999
To study RAG2 gene regulation in vivo, we developed a blastocyst complementation method in which ... more To study RAG2 gene regulation in vivo, we developed a blastocyst complementation method in which RAG2-deficient embryonic stem cells were transfected with genomic clones containing RAG2 and then assessed for their ability to generate lymphocytes. A RAG2 genomic clone that contained only the RAG2 promoter sequences rescued V(D)J recombination in RAG2-deficient pro-B cell lines, but did not rescue development of RAG2-deficient lymphocytes in vivo. However, inclusion of varying lengths of sequences 5 of the RAG2 promoter generated constructs capable of rescuing only in vivo B cell development, as well as other constructs that rescued both B and T cell development. In particular, the 2-kb 5 region starting just upstream of the RAG2 promoter, as well as the region from 2-7 kb 5, could independently drive B cell development, but not efficient T cell development. Deletion of the 2-kb 5 region from the murine germ line demonstrated that this region was not required for RAG expression sufficient to generate normal B or T cell numbers, implying redundancy among 5 elements. We conclude that RAG2 expression in vivo requires elements beyond the core promoter, that such elements contribute to differential regulation in the B vs. T lineages, and that sequences sufficient to direct B cell expression are located in the promoter-proximal 5 region.
TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the tri... more TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the trimeric death receptors (DR4 or DR5). Death receptors are attractive therapeutic targets through both the recombinant TRAIL ligand as well as receptor agonist monoclonal antibodies. Although efficacy of the ligand is hampered by its short half-life, agonistic antibodies have a much longer half-life and have shown some clinical efficacy as antitumor agents. However, the efficacy of these antibodies may be limited by their bivalent nature that does not optimally mimic the trimeric ligand. To overcome limitations of currently used death receptor-targeting agents, we engineered trimeric proteins called Atrimer complexes that selectively bind DR4 and potently induce apoptosis in a variety of cancer cells. Atrimer complexes are based on human tetranectin, a trimeric plasma protein of approximately 60 kDa. Loop regions within the tetranectin C-type lectin domains (CTLD) were randomized to create a large phage display library that was used to select DR4-binding complexes. A panel of unique and potent agonist DR4 Atrimer complexes with subnanomolar affinity to DR4 and no detectable binding to DR5 or the decoy receptors was identified. Mechanism of action studies with a selected Atrimer complex, 1G 2 , showed that Atrimer complexes induce caspase-dependent and DR4-specific apoptosis in cancer cells while sparing normal human fibroblasts and, importantly, hepatocytes. This proof-of-principle study supports the use of alternative proteins engineered to overcome limitations of therapeutically desirable molecules such as TRAIL. Mol Cancer Ther; 11(10); 2087-95. Ó2012 AACR.
To elucidate the intracellular pathways that mediate early B cell development, we directed expres... more To elucidate the intracellular pathways that mediate early B cell development, we directed expression of activated Ras to the B cell lineage in the context of the recombination-activating gene 1 (RAG1)-deficient background (referred to as Ras–RAG). Similar to the effects of an immunoglobulin (Ig) μ heavy chain (HC) transgene, activated Ras caused progression of RAG1–deficient progenitor (pro)-B cells to cells that shared many characteristics with precursor (pre)-B cells, including downregulation of surface CD43 expression plus expression of λ5, RAG2, and germline κ locus transcripts. However, these Ras–RAG pre-B cells also upregulated surface markers characteristic of more mature B cell stages and populated peripheral lymphoid tissues, with an overall phenotype reminiscent of B lineage cells generated in a RAG- deficient background as a result of expression of an Ig μ HC together with a Bcl-2 transgene. Taken together, these findings suggest that activated Ras signaling in pro-B cel...
The 40-kb region downstream of the most 3′ immunoglobulin (Ig) heavy chain constant region gene (... more The 40-kb region downstream of the most 3′ immunoglobulin (Ig) heavy chain constant region gene (Cα) contains a series of transcriptional enhancers speculated to play a role in Ig heavy chain class switch recombination (CSR). To elucidate the function of this putative CSR regulatory region, we generated mice with germline mutations in which one or the other of the two most 5′ enhancers in this cluster (respectively referred to as HS3a and HS1,2) were replaced either with a pgk-neor cassette (referred to as HS3aN and HS1,2N mutations) or with a loxP sequence (referred to as HS3aΔ and HS1,2Δ, respectively). B cells homozygous for the HS3aN or HS1,2N mutations had severe defects in CSR to several isotypes. The phenotypic similarity of the two insertion mutations, both of which were cis-acting, suggested that inhibition might result from pgk-neor cassette gene insertion rather than enhancer deletion. Accordingly, CSR returned to normal in B cells homozygous for the HS3aΔ or HS1,2Δ mutat...
Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process th... more Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process that involves intrachromosomal DNA rearrangement. Ku70 and Ku80 form a DNA end-binding complex required for DNA double strand break repair and V(D)J recombination. Ku70−/− (K70T) mice, like recombination activating gene (RAG)-1– or RAG-2–deficient (R1T or R2T) mice, have impaired B and T cell development at an early progenitor stage, which is thought to result at least in part from defective V(D)J recombination (Gu, Y., K.J. Seidl, G.A. Rathbun, C. Zhu, J.P. Manis, N. van der Stoep, L. Davidson, H.L. Cheng, J.M. Sekiguchi, K. Frank, et al. 1997. Immunity. 7:653–665; Ouyang, H., A. Nussenzweig, A. Kurimasa, V.C. Soares, X. Li, C. Cordon-Cardo, W. Li, N. Cheong, M. Nussenzweig, G. Iliakis, et al. 1997. J. Exp. Med. 186:921–929). Therefore, to examine the potential role of Ku70 in CSR, we generated K70T mice that carry a germline Ig HC locus in which the JH region was replaced with a function...
SAPK is a member of the group of evolutionary conserved stress-activated kinases that mediate con... more SAPK is a member of the group of evolutionary conserved stress-activated kinases that mediate control of cellular death and proliferation. In lymphocytes, the SAPK pathway has been implicated in signaling from antigen, costimulatory, and death receptors; SEK1, which directly activates SAPK, is required for early embryonic development and has also been reported to be essential for normal lymphocyte development. In contrast to the latter findings, we have used RAG-2-deficient blastocyst complementation to show that SEK1-deficient embryonic stem cells support unimpaired T and B lymphocyte development. Moreover, mature SEK1-deficient lymphocytes are capable of SAPK activation. Surprisingly, however, aging SEK1-deficient chimeric mice frequently develop lymphadenopathy and polyclonal B and T cell expansions. Thus, SEK1 is not required for lymphocyte development, but is required for maintaining peripheral lymphoid homeostasis.
Howard Hughes Medical Institute reviewed by Fehling and von Boehmer, 1997). TCR Children's Hospi... more Howard Hughes Medical Institute reviewed by Fehling and von Boehmer, 1997). TCR Children's Hospital variable region genes undergo ordered assembly, with and Department of Genetics DJ rearrangements preceding complete VDJ re-Harvard Medical School arrangements, whereas TCR␦ variable region gene asand The Center for Blood Research sembly is unordered, with DJ␦, VD␦, and DD␦ re-Boston, Massachusetts 02115 arrangements serving as intermediates for complete VDJ␦ rearrangements (Chien et al., 1987b; Migone et al., 1995). In-frame VDJ␦ and VJ␥ rearrangements lead Summary to the expression of a ␥␦ TCR and differentiation along a ␥␦ T cell lineage pathway. Productive VDJ rearrange-We have used gene-targeted mutation to assess the ment leads to the expression of a TCR chain in conjuncrole of the T cell receptor ␦ (TCR␦) enhancer (E␦) in tion with the pre-T␣ protein, differentiation along an ␣ ␣ and ␥␦ T cell development. Mice lacking E␦ exhib-T cell lineage pathway, and transition to the CD4 ϩ CD8 ϩ ited no defects in ␣ T cell development but had a (DP, double positive) stage where germline transcription severe reduction in thymic and peripheral ␥␦ T cells and rearrangement of TCR␣ genes are initiated (reand decreased VDJ␦ rearrangements. Simultaneous viewed by Fehling and von Boehmer, 1997). Once DP deletion of both E␦ and the TCR␣ enhancer (E␣) demthymocytes express an ␣ TCR and successfully unonstrated that residual TCR␦ rearrangements were not dergo positive and negative selection, they differentiate driven by E␣, implicating additional elements in TCR␦ to mature CD4 ϩ CD8 Ϫ or CD4 Ϫ CD8 ϩ (SP, single positive) locus accessibility. Surprisingly, while deletion of E␦ thymocytes (reviewed by Willerford et al., 1996). severely impaired germline TCR␦ expression in dou-The genes that encode the TCR and-␥ chains lie in ble-negative thymocytes, absence of E␦ did not affect distinct loci; whereas the genes that encode the TCR␣ expression of mature ␦ transcripts in ␥␦ T cells. We and-␦ chains lie in a single locus, the TCR␣/␦ locus (see conclude that E␦ has an important role in TCR␦ locus Figure 1A) (Chien et al., 1987a; Satyanarayana et al., regulation at early, but not late, stages of ␥␦ T cell 1988). The V␣ and V␦ gene segments lie in the 5Ј region development. of the locus, with most of the V␦ gene segments located 3Ј of the V␣ gene segments (Wang et al., 1994). The D␦ * To whom correspondence should be addressed (e-mail: alt@ accessibility in ␣ lineages but not ␥␦ lineages, consisrascal.med.harvard.edu).
ABSTRACT We have used gene targeted mutational approaches to assess the role of the T cell recept... more ABSTRACT We have used gene targeted mutational approaches to assess the role of the T cell receptor α (TCRα) enhancer (Eα) in the control of TCRα and TCRδ gene rearrangement and expression. We show that Eα functions in cis to promote Vα to Jα rearrangement across the entire Jα locus, a distance of greater than 70 kb. We also show that Eα is required for normal αβ T cell development; in this lineage, Eα is required for germline Jα expression, for normal expression levels of rearranged VαJα genes, and for expression of a diverse Vα repertoire. In γδ T cells, Eα is not required for VδDJδ rearrangement, but, surprisingly, is required for normal expression levels of mature VδDJδ transcripts and for expression of germline Jα transcripts. Our findings imply that Eα function is not limited to the TCRα components of the TCRα/δ locus or to the αβ lineage; rather, Eα function is important in both αβ and γδ lineage T cells.
The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations ... more The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations in a gene (WASP) encoding a cytoplasmic protein implicated in regulating the actin cytoskeleton. To elucidate WASP function, we disrupted the WASP gene in mice by gene-targeted mutation. WASP-deficient mice showed apparently normal lymphocyte development, normal serum immunoglobulin levels, and the capacity to respond to both T-dependent and T-independent type II antigens. However, these mice did have decreased peripheral blood lymphocyte and platelet numbers and developed chronic colitis. Moreover, purified WASP-deficient T cells showed markedly impaired proliferation and antigen receptor cap formation in response to anti-CD3epsilon stimulation. Yet, purified WASP-deficient B cells showed normal responses to anti-Ig stimulation. We discuss the implications of our findings regarding WASP function in receptor signaling and cytoskeletal reorganization in T and B cells and compare the effects of WASP deficiency in mice and humans.
A central issue in understanding the hematolymphoid system is the generation of appropriate mutan... more A central issue in understanding the hematolymphoid system is the generation of appropriate mutant alleles in mice to reveal the function of regulatory genes. Here we describe a mouse strain, Plastic, with a point mutation in a zinc finger of Ikaros that disrupts DNA binding but preserves efficient assembly of the full-length protein into higher order complexes. Ikaros(Plastic) homozygosity is embryonically lethal with severe defects in terminal erythrocyte and granulocyte differentiation, excessive macrophage formation, and blocked lymphopoiesis, while heterozygotes display a partial block in lymphocyte differentiation. The contrast with more circumscribed effects of Ikaros alleles that ablate the full-length protein highlights the importance in mammals of generating recessive niche-filling alleles that inactivate function without creating a void in multimolecular assemblies.
Ikaros is a unique regulator of lymphopoiesis that associates with pericentromeric heterochromati... more Ikaros is a unique regulator of lymphopoiesis that associates with pericentromeric heterochromatin and has been implicated in heritable gene inactivation. Binding and competition experiments demonstrate that Ikaros dimers compete with an Ets activator for occupancy of the lymphocyte-specific TdT promoter. Mutations that selectively disrupt Ikaros binding to an integrated TdT promoter had no effect on promoter function in a CD4 + CD8 + thymocyte line. However, these mutations abolished down-regulation on differentiation, providing evidence that Ikaros plays a direct role in repression. Reduced access to restriction enzyme cleavage suggested that chromatin alterations accompany down-regulation. The Ikaros-dependent down-regulation event and the observed chromatin alterations appear to precede pericentromeric repositioning. Current models propose that the functions of Ikaros should be disrupted by a small isoform that retains the dimerization domain and lacks the DNA-binding domain. Surprisingly, in the CD4 + CD8 + thymocyte line, overexpression of a small Ikaros isoform had no effect on differentiation or on the pericentromeric targeting and DNA-binding properties of Ikaros. Rather, the small isoform assembled into multimeric complexes with DNA-bound Ikaros at the pericentromeric foci. The capacity for in vivo multimer formation suggests that interactions between Ikaros dimers bound to the TdT promoter and those bound to pericentromeric repeat sequences may contribute to the pericentromeric repositioning of the inactive gene.
Many nuclear proteins are inactivated during mitotic entry, presumably as a prerequisite to chrom... more Many nuclear proteins are inactivated during mitotic entry, presumably as a prerequisite to chromatin condensation and cell division. C2H2 zinc fingers define the largest transcription factor family in the human proteome. The linker separating finger motifs is highly conserved and resembles TGEKP in more than 5000 occurrences. However, the reason for this conservation is not fully understood. We demonstrate that all three linkers in the DNA-binding domain of Ikaros are phosphorylated during mitosis. Phosphomimetic substitutions abolished DNA-binding and pericentromeric localization. A linker within Sp1 was also phosphorylated, suggesting that linker phosphorylation provides a global mechanism for inactivation of the C2H2 family.
Efficient repair of DNA double-strand breaks (DSBs) is Chengming Zhu, 1 Jayanta Chaudhuri, 1 cruc... more Efficient repair of DNA double-strand breaks (DSBs) is Chengming Zhu, 1 Jayanta Chaudhuri, 1 crucial for maintenance of genomic integrity. If unre-Laurie Davidson, 1 Roger Ferrini, 1 paired, such lesions may be cell lethal (Chu, 1997). DSBs Thomas Stamato, 6 Stuart H. Orkin, 3 can be induced in all mammalian cell types during oxida-Michael E. Greenberg, 2 and Frederick W. Alt 1,7 tive metabolism and by genotoxic agents such as ioniz-1 Howard Hughes Medical Institute ing radiation (IR). DSBs are also created in germ cells The Children's Hospital during meiotic recombination, in B and T lymphocytes The Center for Blood Research and during V(D)J recombination (Chu, 1997), and in B lym-Department of Genetics phocytes, possibly, during class switch recombination Harvard University Medical School (CSR) (Wuerffel et al., 1997). In mammalian cells, repair Boston, Massachusetts 02115 of DSBs (DSBR) is achieved by both nonhomologous 2 Department of Neurology end joining (NHEJ) and homologous recombination re-Children's Hospital pair pathways. Defects in the NHEJ pathway lead to Boston, Massachusetts 02115 severe consequences in cells, ranging from increased 3 Howard Hughes Medical Institute IR sensitivity to premature senescence (Smider and Chu, Department of Pediatrics 1997). The importance of the NHEJ pathway is particu-The Children's Hospital larly evident in lymphocytes, as mutations that inactivate Boston, Massachusetts 02115 its components cause premature death of cells at-4 Medical Institute of Bioregulation tempting to undergo V(D)J recombination and CSR (Chang Kyushu University et al., 1995; Casellas et al., 1998; Manis et al., 1998). Maidashi 3-1-1 Much recent knowledge of the functions of NHEJ Higashi-ku, Fukuoka 812 components in DSBR derives from V(D)J recombination Japan studies. The site-specific V(D)J recombination reaction 5 Department of Pathology is targeted by conserved recombination signal se-Tufts University Schools of Medicine quences (RS) that flank each immunoglobulin (Ig) or T and Veterinary Medicine cell receptor (TCR) germline V, D, and J coding segment. Boston, Massachusetts 02111 The lymphocyte-specific RAG1 and RAG2 proteins initi-6 Lankenau Medical Research Center ate the reaction by introducing DSBs between coding 100 Lancaster Avenue segments and flanking RS, resulting in blunt 5Ј-phos-Wynnewood, Pennsylvania 19096 phorylated RS ends and hairpin coding ends (Gellert, 1997). Subsequently, V(D)J recombination becomes a DSBR reaction that employs several generally expressed Summary proteins, initially identified based on studies of IR-sensitive cell lines and mice homozygous for the scid muta-XRCC4 was identified via a complementation cloning tion (SCID mice) (Smider and Chu, 1997). These "DSBR/ method that employed an ionizing radiation (IR)-sensi-V(D)J factors" include Ku80 and DNA-PKcs, two of the tive hamster cell line. By gene-targeted mutation, we three subunits of the DNA-dependent protein kinase show that XRCC4 deficiency in primary murine cells (DNA-PK) holoenzyme, as well as the XRCC4 protein (Li causes growth defects, premature senescence, IR et al., 1995). The roles of two additional DSBR/V(D)J sensitivity, and inability to support V(D)J recombinafactors, Ku70 and ligase IV, implicated based on assocition. In mice, XRCC4 deficiency causes late embryonic ation with Ku80 and XRCC4, respectively (Errami et al., lethality accompanied by defective lymphogenesis 1996; Critchlow et al., 1997; Grawunder et al., 1997), and defective neurogenesis manifested by extensive were demonstrated by gene-targeted mutation studies apoptotic death of newly generated postmitotic neu-(Gu et al., 1997b; Frank et al., 1998; Grawunder et al., ronal cells. We find similar neuronal developmental 1998). defects in embryos that lack DNA ligase IV, an XRCC4-The DNA-PK holoenzyme is comprised of the catalytic associated protein. Our findings demonstrate that difsubunit (DNA-PKcs) and Ku, a heterodimer of Ku70 and ferentiating lymphocytes and neurons strictly require Ku80; Ku binds to DSBs leading to activation of DNAthe XRCC4 and DNA ligase IV end-joining proteins and PKcs. Ku70-and Ku80-deficient mice are viable, although small and immunodeficient; in addition, their cells have defects in DNA end joining, which manifest
Poster Session-Drug targets which provides a Dolichol Phosphate (DolP) substitute on regulation o... more Poster Session-Drug targets which provides a Dolichol Phosphate (DolP) substitute on regulation of Pgp expression in Doxorubicin resistant MCF-7 breast cancer cells. Methods: Breast cancer cell lines, MCF-7 and MCF-7/ADR were used. Pol concentration in the culture medium made up 10 −2-10 −6. Immunohistochemical and Western blotting methods were used to detect the changes in the expression levels of MDR1 and DPAGT1 expression. Intermediates of DPC fractions were analysed by HPLC method. Results: Overexpression of DPAGT1 was detected MCF-7/ADR cells, but not in MCF-7 cells. It is confirmed that plasmatic membranes of MCF-7 cells contain 5.6-6.4% of Pgp (the total protein amount) as a resistance marker. Resistant MCF-7/ADR cells differ from sensitive ones MCF-7 in Pgp content by 10−12 times. The study showed 8.5-fold DolP decrease in MCF-7/ADR cells. The investigations demonstrate that the situation can be changed by treatment with DolP and PP. The DolP concentration in MCF-7/ADR cells was returned to the normal level. It is established that DolP in the concentration 10 −6 M aid 7−9-fold reducing Pgp in membranes of MCF-7/ADR cells. The MCF-7/ADR cells cultivation in medium with polyprenol proceeded to give lowered Pgp content in membranes no over 0.4−0.6%, which amount was consistent with the level of Pgp in MCF-7 cells. Overexpression of DPAGT1 was detected in MCF-7 and in MCF-7/ADR cells. It is established that Pol in the concentration 10 −4 M aid 7−9-fold could overcome DPAGT1 overexpression which leads to regulation of Pgp N-glycosylation. Pol in concentration 10 −2 −10 −3 M induced apoptosis in MCF-7/ADR cells within 3−4 hours. Conclusions: These results indicate that noncontrollable accumulation of Pgp, after MDR1 expression in MCF-7/ADR cells can be overcome using stimulation with dolichyl phosphate substitution. DPAGT1 overexpression in MCF-7/ADR can be overcome with Pol, which provides a DolP substitute for DPAGT1 normal expression.
We have used gene targeted mutational approaches to assess the role of the T cell receptor alpha ... more We have used gene targeted mutational approaches to assess the role of the T cell receptor alpha (TCR alpha) enhancer (E alpha) in the control of TCR alpha and TCR delta gene rearrangement and expression. We show that E alpha functions in cis to promote V alpha to J alpha rearrangement across the entire J alpha locus, a distance of greater than 70 kb. We also show that E alpha is required for normal alphabeta T cell development; in this lineage, E alpha is required for germline J alpha expression, for normal expression levels of rearranged V alpha J alpha genes, and for expression of a diverse V alpha repertoire. In gamma delta T cells, E alpha is not required for VdeltaDJdelta rearrangement, but, surprisingly, is required for normal expression levels of mature VdeltaDJdelta transcripts and for expression of germline J alpha transcripts. Our findings imply that E alpha function is not limited to the TCR alpha components of the TCRalpha/delta locus or to the alpha beta lineage; rathe...
Summary The 40-kb region downstream of the most 3 9 immunoglobulin (Ig) heavy chain constant re- ... more Summary The 40-kb region downstream of the most 3 9 immunoglobulin (Ig) heavy chain constant re- gion gene (C a ) contains a series of transcriptional enhancers speculated to play a role in Ig heavy chain class switch recombination (CSR). To elucidate the function of this putative CSR regulatory region, we generated mice with germline mutations in which one or the other of the two most 5 9 enhancers in this cluster (respectively referred to as HS3a and HS1,2) were re- placed either with a pgk- neo r cassette (referred to as HS3aN and HS1,2N mutations) or with a loxP sequence (referred to as HS3a D and HS1,2 D , respectively). B cells homozygous for the HS3aN or HS1,2N mutations had severe defects in CSR to several isotypes. The phenotypic similarity of the two insertion mutations, both of which were cis -acting, suggested that inhibi- tion might result from pgk- neo r cassette gene insertion rather than enhancer deletion. Accord- ingly, CSR returned to normal in B cells homozygous f...
Proceedings of the National Academy of Sciences, 1999
To study RAG2 gene regulation in vivo, we developed a blastocyst complementation method in which ... more To study RAG2 gene regulation in vivo, we developed a blastocyst complementation method in which RAG2-deficient embryonic stem cells were transfected with genomic clones containing RAG2 and then assessed for their ability to generate lymphocytes. A RAG2 genomic clone that contained only the RAG2 promoter sequences rescued V(D)J recombination in RAG2-deficient pro-B cell lines, but did not rescue development of RAG2-deficient lymphocytes in vivo. However, inclusion of varying lengths of sequences 5 of the RAG2 promoter generated constructs capable of rescuing only in vivo B cell development, as well as other constructs that rescued both B and T cell development. In particular, the 2-kb 5 region starting just upstream of the RAG2 promoter, as well as the region from 2-7 kb 5, could independently drive B cell development, but not efficient T cell development. Deletion of the 2-kb 5 region from the murine germ line demonstrated that this region was not required for RAG expression sufficient to generate normal B or T cell numbers, implying redundancy among 5 elements. We conclude that RAG2 expression in vivo requires elements beyond the core promoter, that such elements contribute to differential regulation in the B vs. T lineages, and that sequences sufficient to direct B cell expression are located in the promoter-proximal 5 region.
TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the tri... more TRAIL is a trimeric protein that potently induces apoptosis in cancer cells by binding to the trimeric death receptors (DR4 or DR5). Death receptors are attractive therapeutic targets through both the recombinant TRAIL ligand as well as receptor agonist monoclonal antibodies. Although efficacy of the ligand is hampered by its short half-life, agonistic antibodies have a much longer half-life and have shown some clinical efficacy as antitumor agents. However, the efficacy of these antibodies may be limited by their bivalent nature that does not optimally mimic the trimeric ligand. To overcome limitations of currently used death receptor-targeting agents, we engineered trimeric proteins called Atrimer complexes that selectively bind DR4 and potently induce apoptosis in a variety of cancer cells. Atrimer complexes are based on human tetranectin, a trimeric plasma protein of approximately 60 kDa. Loop regions within the tetranectin C-type lectin domains (CTLD) were randomized to create a large phage display library that was used to select DR4-binding complexes. A panel of unique and potent agonist DR4 Atrimer complexes with subnanomolar affinity to DR4 and no detectable binding to DR5 or the decoy receptors was identified. Mechanism of action studies with a selected Atrimer complex, 1G 2 , showed that Atrimer complexes induce caspase-dependent and DR4-specific apoptosis in cancer cells while sparing normal human fibroblasts and, importantly, hepatocytes. This proof-of-principle study supports the use of alternative proteins engineered to overcome limitations of therapeutically desirable molecules such as TRAIL. Mol Cancer Ther; 11(10); 2087-95. Ó2012 AACR.
To elucidate the intracellular pathways that mediate early B cell development, we directed expres... more To elucidate the intracellular pathways that mediate early B cell development, we directed expression of activated Ras to the B cell lineage in the context of the recombination-activating gene 1 (RAG1)-deficient background (referred to as Ras–RAG). Similar to the effects of an immunoglobulin (Ig) μ heavy chain (HC) transgene, activated Ras caused progression of RAG1–deficient progenitor (pro)-B cells to cells that shared many characteristics with precursor (pre)-B cells, including downregulation of surface CD43 expression plus expression of λ5, RAG2, and germline κ locus transcripts. However, these Ras–RAG pre-B cells also upregulated surface markers characteristic of more mature B cell stages and populated peripheral lymphoid tissues, with an overall phenotype reminiscent of B lineage cells generated in a RAG- deficient background as a result of expression of an Ig μ HC together with a Bcl-2 transgene. Taken together, these findings suggest that activated Ras signaling in pro-B cel...
The 40-kb region downstream of the most 3′ immunoglobulin (Ig) heavy chain constant region gene (... more The 40-kb region downstream of the most 3′ immunoglobulin (Ig) heavy chain constant region gene (Cα) contains a series of transcriptional enhancers speculated to play a role in Ig heavy chain class switch recombination (CSR). To elucidate the function of this putative CSR regulatory region, we generated mice with germline mutations in which one or the other of the two most 5′ enhancers in this cluster (respectively referred to as HS3a and HS1,2) were replaced either with a pgk-neor cassette (referred to as HS3aN and HS1,2N mutations) or with a loxP sequence (referred to as HS3aΔ and HS1,2Δ, respectively). B cells homozygous for the HS3aN or HS1,2N mutations had severe defects in CSR to several isotypes. The phenotypic similarity of the two insertion mutations, both of which were cis-acting, suggested that inhibition might result from pgk-neor cassette gene insertion rather than enhancer deletion. Accordingly, CSR returned to normal in B cells homozygous for the HS3aΔ or HS1,2Δ mutat...
Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process th... more Immunoglobulin (Ig) heavy chain (HC) class switch recombination (CSR) is a late B cell process that involves intrachromosomal DNA rearrangement. Ku70 and Ku80 form a DNA end-binding complex required for DNA double strand break repair and V(D)J recombination. Ku70−/− (K70T) mice, like recombination activating gene (RAG)-1– or RAG-2–deficient (R1T or R2T) mice, have impaired B and T cell development at an early progenitor stage, which is thought to result at least in part from defective V(D)J recombination (Gu, Y., K.J. Seidl, G.A. Rathbun, C. Zhu, J.P. Manis, N. van der Stoep, L. Davidson, H.L. Cheng, J.M. Sekiguchi, K. Frank, et al. 1997. Immunity. 7:653–665; Ouyang, H., A. Nussenzweig, A. Kurimasa, V.C. Soares, X. Li, C. Cordon-Cardo, W. Li, N. Cheong, M. Nussenzweig, G. Iliakis, et al. 1997. J. Exp. Med. 186:921–929). Therefore, to examine the potential role of Ku70 in CSR, we generated K70T mice that carry a germline Ig HC locus in which the JH region was replaced with a function...
SAPK is a member of the group of evolutionary conserved stress-activated kinases that mediate con... more SAPK is a member of the group of evolutionary conserved stress-activated kinases that mediate control of cellular death and proliferation. In lymphocytes, the SAPK pathway has been implicated in signaling from antigen, costimulatory, and death receptors; SEK1, which directly activates SAPK, is required for early embryonic development and has also been reported to be essential for normal lymphocyte development. In contrast to the latter findings, we have used RAG-2-deficient blastocyst complementation to show that SEK1-deficient embryonic stem cells support unimpaired T and B lymphocyte development. Moreover, mature SEK1-deficient lymphocytes are capable of SAPK activation. Surprisingly, however, aging SEK1-deficient chimeric mice frequently develop lymphadenopathy and polyclonal B and T cell expansions. Thus, SEK1 is not required for lymphocyte development, but is required for maintaining peripheral lymphoid homeostasis.
Howard Hughes Medical Institute reviewed by Fehling and von Boehmer, 1997). TCR Children's Hospi... more Howard Hughes Medical Institute reviewed by Fehling and von Boehmer, 1997). TCR Children's Hospital variable region genes undergo ordered assembly, with and Department of Genetics DJ rearrangements preceding complete VDJ re-Harvard Medical School arrangements, whereas TCR␦ variable region gene asand The Center for Blood Research sembly is unordered, with DJ␦, VD␦, and DD␦ re-Boston, Massachusetts 02115 arrangements serving as intermediates for complete VDJ␦ rearrangements (Chien et al., 1987b; Migone et al., 1995). In-frame VDJ␦ and VJ␥ rearrangements lead Summary to the expression of a ␥␦ TCR and differentiation along a ␥␦ T cell lineage pathway. Productive VDJ rearrange-We have used gene-targeted mutation to assess the ment leads to the expression of a TCR chain in conjuncrole of the T cell receptor ␦ (TCR␦) enhancer (E␦) in tion with the pre-T␣ protein, differentiation along an ␣ ␣ and ␥␦ T cell development. Mice lacking E␦ exhib-T cell lineage pathway, and transition to the CD4 ϩ CD8 ϩ ited no defects in ␣ T cell development but had a (DP, double positive) stage where germline transcription severe reduction in thymic and peripheral ␥␦ T cells and rearrangement of TCR␣ genes are initiated (reand decreased VDJ␦ rearrangements. Simultaneous viewed by Fehling and von Boehmer, 1997). Once DP deletion of both E␦ and the TCR␣ enhancer (E␣) demthymocytes express an ␣ TCR and successfully unonstrated that residual TCR␦ rearrangements were not dergo positive and negative selection, they differentiate driven by E␣, implicating additional elements in TCR␦ to mature CD4 ϩ CD8 Ϫ or CD4 Ϫ CD8 ϩ (SP, single positive) locus accessibility. Surprisingly, while deletion of E␦ thymocytes (reviewed by Willerford et al., 1996). severely impaired germline TCR␦ expression in dou-The genes that encode the TCR and-␥ chains lie in ble-negative thymocytes, absence of E␦ did not affect distinct loci; whereas the genes that encode the TCR␣ expression of mature ␦ transcripts in ␥␦ T cells. We and-␦ chains lie in a single locus, the TCR␣/␦ locus (see conclude that E␦ has an important role in TCR␦ locus Figure 1A) (Chien et al., 1987a; Satyanarayana et al., regulation at early, but not late, stages of ␥␦ T cell 1988). The V␣ and V␦ gene segments lie in the 5Ј region development. of the locus, with most of the V␦ gene segments located 3Ј of the V␣ gene segments (Wang et al., 1994). The D␦ * To whom correspondence should be addressed (e-mail: alt@ accessibility in ␣ lineages but not ␥␦ lineages, consisrascal.med.harvard.edu).
ABSTRACT We have used gene targeted mutational approaches to assess the role of the T cell recept... more ABSTRACT We have used gene targeted mutational approaches to assess the role of the T cell receptor α (TCRα) enhancer (Eα) in the control of TCRα and TCRδ gene rearrangement and expression. We show that Eα functions in cis to promote Vα to Jα rearrangement across the entire Jα locus, a distance of greater than 70 kb. We also show that Eα is required for normal αβ T cell development; in this lineage, Eα is required for germline Jα expression, for normal expression levels of rearranged VαJα genes, and for expression of a diverse Vα repertoire. In γδ T cells, Eα is not required for VδDJδ rearrangement, but, surprisingly, is required for normal expression levels of mature VδDJδ transcripts and for expression of germline Jα transcripts. Our findings imply that Eα function is not limited to the TCRα components of the TCRα/δ locus or to the αβ lineage; rather, Eα function is important in both αβ and γδ lineage T cells.
The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations ... more The Wiskott-Aldrich syndrome (WAS) is a human X-linked immunodeficiency resulting from mutations in a gene (WASP) encoding a cytoplasmic protein implicated in regulating the actin cytoskeleton. To elucidate WASP function, we disrupted the WASP gene in mice by gene-targeted mutation. WASP-deficient mice showed apparently normal lymphocyte development, normal serum immunoglobulin levels, and the capacity to respond to both T-dependent and T-independent type II antigens. However, these mice did have decreased peripheral blood lymphocyte and platelet numbers and developed chronic colitis. Moreover, purified WASP-deficient T cells showed markedly impaired proliferation and antigen receptor cap formation in response to anti-CD3epsilon stimulation. Yet, purified WASP-deficient B cells showed normal responses to anti-Ig stimulation. We discuss the implications of our findings regarding WASP function in receptor signaling and cytoskeletal reorganization in T and B cells and compare the effects of WASP deficiency in mice and humans.
A central issue in understanding the hematolymphoid system is the generation of appropriate mutan... more A central issue in understanding the hematolymphoid system is the generation of appropriate mutant alleles in mice to reveal the function of regulatory genes. Here we describe a mouse strain, Plastic, with a point mutation in a zinc finger of Ikaros that disrupts DNA binding but preserves efficient assembly of the full-length protein into higher order complexes. Ikaros(Plastic) homozygosity is embryonically lethal with severe defects in terminal erythrocyte and granulocyte differentiation, excessive macrophage formation, and blocked lymphopoiesis, while heterozygotes display a partial block in lymphocyte differentiation. The contrast with more circumscribed effects of Ikaros alleles that ablate the full-length protein highlights the importance in mammals of generating recessive niche-filling alleles that inactivate function without creating a void in multimolecular assemblies.
Ikaros is a unique regulator of lymphopoiesis that associates with pericentromeric heterochromati... more Ikaros is a unique regulator of lymphopoiesis that associates with pericentromeric heterochromatin and has been implicated in heritable gene inactivation. Binding and competition experiments demonstrate that Ikaros dimers compete with an Ets activator for occupancy of the lymphocyte-specific TdT promoter. Mutations that selectively disrupt Ikaros binding to an integrated TdT promoter had no effect on promoter function in a CD4 + CD8 + thymocyte line. However, these mutations abolished down-regulation on differentiation, providing evidence that Ikaros plays a direct role in repression. Reduced access to restriction enzyme cleavage suggested that chromatin alterations accompany down-regulation. The Ikaros-dependent down-regulation event and the observed chromatin alterations appear to precede pericentromeric repositioning. Current models propose that the functions of Ikaros should be disrupted by a small isoform that retains the dimerization domain and lacks the DNA-binding domain. Surprisingly, in the CD4 + CD8 + thymocyte line, overexpression of a small Ikaros isoform had no effect on differentiation or on the pericentromeric targeting and DNA-binding properties of Ikaros. Rather, the small isoform assembled into multimeric complexes with DNA-bound Ikaros at the pericentromeric foci. The capacity for in vivo multimer formation suggests that interactions between Ikaros dimers bound to the TdT promoter and those bound to pericentromeric repeat sequences may contribute to the pericentromeric repositioning of the inactive gene.
Many nuclear proteins are inactivated during mitotic entry, presumably as a prerequisite to chrom... more Many nuclear proteins are inactivated during mitotic entry, presumably as a prerequisite to chromatin condensation and cell division. C2H2 zinc fingers define the largest transcription factor family in the human proteome. The linker separating finger motifs is highly conserved and resembles TGEKP in more than 5000 occurrences. However, the reason for this conservation is not fully understood. We demonstrate that all three linkers in the DNA-binding domain of Ikaros are phosphorylated during mitosis. Phosphomimetic substitutions abolished DNA-binding and pericentromeric localization. A linker within Sp1 was also phosphorylated, suggesting that linker phosphorylation provides a global mechanism for inactivation of the C2H2 family.
Efficient repair of DNA double-strand breaks (DSBs) is Chengming Zhu, 1 Jayanta Chaudhuri, 1 cruc... more Efficient repair of DNA double-strand breaks (DSBs) is Chengming Zhu, 1 Jayanta Chaudhuri, 1 crucial for maintenance of genomic integrity. If unre-Laurie Davidson, 1 Roger Ferrini, 1 paired, such lesions may be cell lethal (Chu, 1997). DSBs Thomas Stamato, 6 Stuart H. Orkin, 3 can be induced in all mammalian cell types during oxida-Michael E. Greenberg, 2 and Frederick W. Alt 1,7 tive metabolism and by genotoxic agents such as ioniz-1 Howard Hughes Medical Institute ing radiation (IR). DSBs are also created in germ cells The Children's Hospital during meiotic recombination, in B and T lymphocytes The Center for Blood Research and during V(D)J recombination (Chu, 1997), and in B lym-Department of Genetics phocytes, possibly, during class switch recombination Harvard University Medical School (CSR) (Wuerffel et al., 1997). In mammalian cells, repair Boston, Massachusetts 02115 of DSBs (DSBR) is achieved by both nonhomologous 2 Department of Neurology end joining (NHEJ) and homologous recombination re-Children's Hospital pair pathways. Defects in the NHEJ pathway lead to Boston, Massachusetts 02115 severe consequences in cells, ranging from increased 3 Howard Hughes Medical Institute IR sensitivity to premature senescence (Smider and Chu, Department of Pediatrics 1997). The importance of the NHEJ pathway is particu-The Children's Hospital larly evident in lymphocytes, as mutations that inactivate Boston, Massachusetts 02115 its components cause premature death of cells at-4 Medical Institute of Bioregulation tempting to undergo V(D)J recombination and CSR (Chang Kyushu University et al., 1995; Casellas et al., 1998; Manis et al., 1998). Maidashi 3-1-1 Much recent knowledge of the functions of NHEJ Higashi-ku, Fukuoka 812 components in DSBR derives from V(D)J recombination Japan studies. The site-specific V(D)J recombination reaction 5 Department of Pathology is targeted by conserved recombination signal se-Tufts University Schools of Medicine quences (RS) that flank each immunoglobulin (Ig) or T and Veterinary Medicine cell receptor (TCR) germline V, D, and J coding segment. Boston, Massachusetts 02111 The lymphocyte-specific RAG1 and RAG2 proteins initi-6 Lankenau Medical Research Center ate the reaction by introducing DSBs between coding 100 Lancaster Avenue segments and flanking RS, resulting in blunt 5Ј-phos-Wynnewood, Pennsylvania 19096 phorylated RS ends and hairpin coding ends (Gellert, 1997). Subsequently, V(D)J recombination becomes a DSBR reaction that employs several generally expressed Summary proteins, initially identified based on studies of IR-sensitive cell lines and mice homozygous for the scid muta-XRCC4 was identified via a complementation cloning tion (SCID mice) (Smider and Chu, 1997). These "DSBR/ method that employed an ionizing radiation (IR)-sensi-V(D)J factors" include Ku80 and DNA-PKcs, two of the tive hamster cell line. By gene-targeted mutation, we three subunits of the DNA-dependent protein kinase show that XRCC4 deficiency in primary murine cells (DNA-PK) holoenzyme, as well as the XRCC4 protein (Li causes growth defects, premature senescence, IR et al., 1995). The roles of two additional DSBR/V(D)J sensitivity, and inability to support V(D)J recombinafactors, Ku70 and ligase IV, implicated based on assocition. In mice, XRCC4 deficiency causes late embryonic ation with Ku80 and XRCC4, respectively (Errami et al., lethality accompanied by defective lymphogenesis 1996; Critchlow et al., 1997; Grawunder et al., 1997), and defective neurogenesis manifested by extensive were demonstrated by gene-targeted mutation studies apoptotic death of newly generated postmitotic neu-(Gu et al., 1997b; Frank et al., 1998; Grawunder et al., ronal cells. We find similar neuronal developmental 1998). defects in embryos that lack DNA ligase IV, an XRCC4-The DNA-PK holoenzyme is comprised of the catalytic associated protein. Our findings demonstrate that difsubunit (DNA-PKcs) and Ku, a heterodimer of Ku70 and ferentiating lymphocytes and neurons strictly require Ku80; Ku binds to DSBs leading to activation of DNAthe XRCC4 and DNA ligase IV end-joining proteins and PKcs. Ku70-and Ku80-deficient mice are viable, although small and immunodeficient; in addition, their cells have defects in DNA end joining, which manifest
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