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PubMed1998

Discrete stages of anterior pituitary differentiation recapitulated in immortalized cell lines.

Targeted expression of oncogenes in transgenic mice can immortalize specific cell types to serve as valuable cultured model systems. Utilizing promoter regions from pituitary genes activated in the gonadotrope/thyrotrope lineage at discrete stages of development, we have demonstrated that targeted oncogene expression in transgenic mice can produce cell lines representing sequential stages of differentiation. Each cell line expresses a specific subset of the genes denoting differentiated function such as the subunits of the glycoprotein hormones, hormone receptors, and transcriptional regulatory proteins. These model systems have allowed detailed investigations into molecular and cellular mechanisms otherwise inaccessible in vivo or in complex primary pituitary cell cultures.

Animals↗

PubMed1978

Sensitization of lymphocytes against pooled allogeneic cells. I. Generation of cytotoxicity against autologous human lymphoblastoid cell lines.

Lymphocytes sensitized in vitro to a pool of X-irradiated allogeneic normal lymphocytes from 20 individuals develop cytotoxic activity for autologous human lymphoblastoid cells (LCL). Whereas pool sensitized T lymphocytes lyse autologous LCL cells, they fail to lyse autologous B-enriched or T-enriched normal target cells nor autologous phytohemagglutinin (PHA) blasts. In contrast to pool sensitization, stimulation with normal cells of single allogeneic individuals rarely led to development of cytotoxicity against autologous LCL cells. We conclude that human Epstein-Barr virus transformed LCL cells express target antigens cross-reactive with allogeneic target antigens expressed on normal cells and that sensitization with a pool of allogeneic cells is an effective means of generating effector cells directed against autologous abnormal cells.

B-Lymphocytes↗

PubMed1990

Assessment of the specificity of cytotoxic T lymphocytes for the nucleoprotein of Pichinde virus using recombinant vaccinia viruses.

Pichinde virus (PV) infection of mice results in induction of a strong H-2 restricted, virus-specific cytotoxic T lymphocyte (CTL) response and rapid clearance of the virus. To define the specificities of CTL induced by PV infection, we constructed vaccinia virus recombinants containing cloned cDNAs corresponding to full-length (VVNP) and a truncated form (VVNP 51-561) of the nucleoprotein (NP) gene of PV. Radioimmunoprecipitation analysis of infected cell lysates indicated that VVNP expressed a PV-specific product identical in size to that of authentic NP, while vaccinia virus recombinants containing truncated NP produced a polypeptide consistent with the synthesis of amino acids 51-561 of Pichinde virus NP. Interestingly, cells infected with VVNP synthesized easily detectable, but much lower levels of nucleoprotein relative to both PV and VVNP51-561. Primary virus-specific CTL induced in three different strains of inbred mice following intravenous infection with PV were able to lyse syngeneic target cells infected with PV but did not markedly lyse syngeneic targets expressing full-length or truncated NP following recombinant vaccinia virus infection. Similarly, secondary anti-PV specific CTL generated following in vitro restimulation by PV or selectively restimulated with vaccinia recombinants did not significantly lyse target cells expressing NP. Further, infection of mice with VVNP and VVNP51-561 did not induce CTLs specific for PV and did not prime mice for the generation of memory anti-PV CTL in vivo. These results suggest that PV gene products other than NP, such as the GPC or L protein, contain the major target epitope(s) recognized by PV-specific CTL.

Animals↗

PubMed2026

A single-cell atlas of multiple myeloma defines malignant archetypes and proliferative states.

Multiple myeloma (MM) is a plasma-cell malignancy with extensive genomic and transcriptional heterogeneity, limiting disease classification and precision therapy. Here we generated a clinically annotated, population-scale, single-cell atlas of MM from 341 individuals spanning the disease and treatment continuum. We identified five recurrent malignant transcriptional archetypes and an orthogonal proliferative program associated with genomic features, therapeutic resistance and clinical outcomes. Validation in the independent CoMMpass cohort demonstrated robustness, prognostic relevance and portability across platforms. We developed a single-cell, target-discovery pipeline prioritizing malignant enrichment, cell-type specificity and tissue restriction, identifying FCRL2 as a plasma-restricted or B cell-lineage-restricted surface target expressed by malignant plasma cells. FCRL2-targeted chimeric antigen receptor T cells demonstrated antigen-specific activity in vitro and survival benefit in vivo. Together, these data provide a clinically actionable blueprint for patient stratification and precision target nomination in plasma-cell malignancies.

Multiple Myeloma↗

PubMed1988

Increased resistance to non-MHC-restricted cytotoxicity related to HLA A, B expression. Direct demonstration using beta 2-microglobulin-transfected Daudi cells.

Experiments in several laboratories have shown that target susceptibility to NK and lymphokine-activated killer (LAK) cytotoxicity is inversely correlated with the target expression of HLA Class I molecules. We present the first direct evidence, obtained by gene transfection, that target cell HLA, A, B expression increases the resistance to the "so-called" non-MHC-restricted cytotoxicity. We have co-transfected, by electroporation, the human beta 2-microglobulin gene and the gene carrying the resistance to geneticin into Daudi cell line. Geneticin selection in culture followed by FACS sorting on the basis of strong positivity with the mAb W6/32 (which is specific for the HLA class I H chain associated to beta 2-microglobulin) have led to the establishment of a HLA+ Daudi cell line permanently expressing HLA A10, A11, and B17 molecules. Studies were performed in vitro to evaluate the susceptibility of these cells to either NK and LAK cytotoxicity. The HLA class I+ Daudi cells exhibit an increased resistance to killing by non-MHC-restricted killer cells (both NK and LAK) as compared with their HLA-Daudi counterpart.

Cytotoxicity, Immunologic↗

PubMed1998

Cloning and characterization of Pros45, the Drosophila SUG1 proteasome subunit homolog.

The proteasome plays essential roles in a variety of cellular processes, including degradation of the bulk of cellular proteins, degradation of short-lived proteins such as cell cycle regulators, generation of antigenic peptides, and mediating programmed cell death. One of the best characterized subunits of the 26S proteasome is encoded by the yeast gene SUG1. We report here the cloning and characterization of the Drosophila homolog of this gene, Pros45. At the protein level, Pros45 is highly conserved with respect to its homologs in a variety of taxa: it shows 74% identity to yeast Sug1; 86% to mouse m56/mSug1/FZA-B; 87% to human Trip1; and 97% to moth 18-56. Using a genomic clone as a probe for in situ hyridization to polytene chromesomes, we demonstrated that Pros45 maps to 19F, near the base of the X chromosome. Use of a pros45 cDNA clone as a probe revealed a second site of hybridization at 99CD. Pros45 mRNA is found in the unfertilized egg and in all cells of the early embryo. By the end of embryogenesis, Pros45 is expressed predominantly in the central nervous system. Targeted expression of Pros45 in a variety of different cells using the Gal4 UAS P-element system failed to generate an overt phenotype. This study provides the foundation for further examination of the role of the 26S proteasome in homeostasis and development in Drosophila.

Adenosine Triphosphatases↗

PubMed2002

Advances in lentiviral vector design for gene-modification of hematopoietic stem cells.

Lentiviral vectors are more efficient at transducing quiescent hematopoietic stem cells than murine retroviral vectors. This characteristic is due to multiple karyophilic components of the lentiviral vector pre-integration complex. Lentiviral vectors are also able to carry more complex payloads than murine retroviral vectors, making it possible to deliver expression cassettes that direct either constitutive or targeted expression in various hematopoietic stem cell progeny.

Animals↗

PubMed2002

Carcinoembryonic antigen (CEA) presentation and specific T cell-priming by human dendritic cells transfected with CEA-mRNA.

The feasibility of dendritic cells (DC) for cancer immunotherapy after transfection by electroporation with mRNA encoding the human carcinoembryonic antigen (CEA) was investigated. Both, total RNA from the CEA(+) colon cancer cell line SW480 and mRNA transcribed in vitro from cDNA3.1-plasmids (pcDNA3.1+/-HisC) with a CEA-insert (ivt-CEA-mRNA, ivt-CEA/HisC-mRNA) were used. Labelled ivt-CEA-mRNA was detectable in DC by light and electron microscopy and by fluorescence-activated cell-sorting (FACS) even 15 min after electroporation. Four hours after transfection with ivt-CEA/HisC-mRNA, we detected specific expression of CEA and the histidine-tag by immunofluorescence microscopy and by FACS. CEA-specific T lymphocytes were successfully primed by transfected DC and were able to lyse CEA-expressing target cells, even from the CEA-expressing human colon adenocarcinoma cell line SW480. Thus, DC transfected by electroporation with CEA-mRNA are valuable tools for the immunotherapy of CEA(+) tumour entities.

Antigen Presentation↗

PubMed2002

Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced neurodegeneration.

We have successfully generated a Drosophila model of human polyglutamine (polyQ) diseases by the targeted expression of expanded-polyQ (ex-polyQ) in the Drosophila compound eye. The resulting eye degeneration is progressive and ex-polyQ dosage- and ex-polyQ length-dependent. Furthermore, intergenerational changes in repeat length were observed in homozygotes, with concomitant changes in the levels of degeneration. Through genetic screening, using this fly model, we identified loss-of-function mutants of the ter94 gene that encodes the Drosophila homolog of VCP/CDC48, a member of the AAA+ class of the ATPase protein family, as dominant suppressors. The suppressive effects of the ter94 mutants on ex-polyQ-induced neurodegeneration correlated well with the degrees of loss-of-function, but appeared not to result from the inhibition of ex-polyQ aggregate formation. In the ex-polyQ-expressing cells of the late pupa, an upregulation of ter94 expression was observed prior to cell death. Co-expression of ter94 with ex-polyQ severely enhanced eye degeneration. Interestingly, when ter94 was overexpressed in the eye by increasing the transgene copies, severe eye degeneration was induced. Furthermore, genetical studies revealed that ter94 was not involved in grim-, reaper-, hid-, ced4-, or p53-induced cell death pathways. From these observations, we propose that VCP is a novel cell death effector molecule in ex-polyQ-induced neurodegeneration, where the amount of VCP is critical. Control of VCP expression may thus be a potential therapeutic target in ex-polyQ-induced neurodegeneration.

Adenosine Triphosphatases↗

PubMed1999

Diverse stabilities of expression in the rat brain from different cellular promoters in a helper virus-free herpes simplex virus type 1 vector system.

Many neural gene transfer studies require both long-term and cell type-specific expression. We have reported a helper virus-free HSV-1 plasmid vector system (Fraefel et al., 1996), and this system supports at least some long-term expression from herpesvirus immediate-early promoters. In this study, we constructed vectors that placed the lacZ reporter gene under the regulation of five different cellular promoters. Vector stocks were microinjected into the midbrain, striatum, or hippocampus; the rats were sacrificed at 4 days to 2 months after gene transfer; and the numbers of X-Gal-positive cells were determined. A 6.8-kb fragment of the rat tyrosine hydroxylase (TH) promoter supported relatively stable expression for up to 2 months and targeted expression to TH-immunoreactive neurons in the substantia nigra pars compacta. The other promoters that were examined were chosen with the goal of obtaining long-term, neuronal-specific expression. At 4 days after gene transfer, a 766-bp fragment of the TH promoter supported expression in cells with neuronal morphology in the midbrain and striatum, consistent with results in transgenic mice. However, expression was absent by 2 weeks. Similarly, at 4 days after gene transfer, a mouse neurofilament heavy subunit promoter supported expression in cells with neuronal morphology in the midbrain, striatum, and hippocampus, but expression was absent by 2 weeks. A rat neuron-specific enolase promoter supported only a low level of expression in cultured neuronal cells, and expression was not detected in the brain. A rat voltage-gated sodium channel promoter supported only a low level of expression in PC12 cells and expression could not be detected in cultured cortical cells. These results demonstrate that different promoters support a wide range of levels and stabilities of expression in this vector system, and the results suggest approaches to improving the stability of long-term expression.

Animals↗

PubMed1995

Gonadal tumorigenesis in transgenic mice bearing the mouse inhibin alpha-subunit promoter/simian virus T-antigen fusion gene: characterization of ovarian tumors and establishment of gonadotropin-responsive granulosa cell lines.

To establish in vivo gonadal tumor models and permanent lines of gonadal somatic cells we produced transgenic (TG) mice expressing the Simian virus (SV) 40 T-antigens (T-ag), driven by 6 or 2.1 kilobase fragments of the mouse inhibin alpha-subunit promoter. Hitherto, altogether 44 TG mice, one of which carried the shorter transgene, have produced gonadal tumors. Two founder females expressing the longer transgene, KK1 and KK3, and three established TG mouse lines were studied in detail. Penetrance of the phenotype in IT6-M and IT6-F mouse lines was 100% (tumors/TG: IT6-M 22/22, IT6-F 14/14). The T-ag mRNA was strongly expressed in the gonads, adrenal glands, pituitary, and brain. The KK-1 and KK-3 ovarian tumor cells immunostained with anti-SV40 large-T antibody. The KK-1 cells possessed high-affinity LH receptors [equilibrium association constant (Ka = 7.8 x 10(10) liters/mol] and responded to human CG by elevated cAMP and progesterone production. Also FSH slightly stimulated their cAMP and estradiol production (P < 0.01). These cells expressed cytochrome P450arom and inhibin alpha mRNA, but not cytochrome P450c17 alpha. In conclusion, the KK-1 cells are immortalized luteinizing granulosa cells expressing endogenous gonadotropin receptors, steroidogenic enzymes, and inhibin alpha. These cells will be useful in studies on the molecular aspects of granulosa cell function. The present study indicates that the 6-kilobase fragment of the inhibin alpha promoter described in this article contains the elements directing tissue-specific expression in vivo and is useful for targeted expression of other genes in the gonads.

Activins↗

PubMed1995

wingless is required for the formation of a subset of muscle founder cells during Drosophila embryogenesis.

The final pattern of the Drosophila larval body wall muscles depends critically on the prior segregation of muscle founder cells. We would like to understand the underlying molecular mechanisms which ensure the precise allocation and placement of these muscle founder cells. We have begun our analysis by examining the role of the segment polarity genes, known to be involved in the patterning of the ectoderm. Mutations in only one member of this class, wingless (wg), lead to the complete loss of a subset of muscle founder cells characterised by the expression of S59. Using the GAL4-targetted expression system, we find that Wingless, a secreted glycoprotein and well characterized signalling molecule, acts directly on the mesoderm to ensure the formation of S59-expressing founder cells. Moreover, we present evidence that Wg can signal across germ layers and that, in the wild-type embryo, Wg from the ectoderm could constitute an inductive signal for the initiation of the development of a subset of somatic muscles.

Animals↗

PubMed2000

Design of retroviral vectors and helper cells for gene therapy.

During the past decade, gene therapy has been applied to the treatment of disease in hundreds of clinical trials. Various tools have been developed to deliver genes into human cells; among them, genetically engineered retroviruses are currently the most popular tool for gene delivery. Most of the systems contain vectors that are capable of accommodating genes of interest and helper cells that can provide the viral structural proteins and enzymes to allow for the generation of vector-containing infectious viral particles. Retroviridae is a family of retroviruses that differs in nucleotide and amino acid sequence, genome structure, pathogenicity, and host range. This diversity provides opportunities to use viruses with different biological characteristics to develop different therapeutic applications. Currently, a variety of retroviruses that provide distinct advantages for gene delivery has been modified and used in clinical trials. In this review, the genome structures of oncoviruses, lentiviruses, and spumaviruses are reviewed and examples of vectors derived from these viruses are described. As with any delivery tool, the efficiency, the ability to target certain tissue or cell type, the expression of the gene of interest, and the safety of retroviral-based systems are important for successful application of gene therapy. Significant efforts have been dedicated to these areas of research in recent years. Various modifications have been made to retroviral-based vectors and helper cells to alter gene expression, target delivery, improve viral titers, and increase safety. The principles and design of these modifications are discussed in this review.

Animals↗

PubMed2003

Loss of protein kinase Calpha expression may enhance the tumorigenic potential of Gli1 in basal cell carcinoma.

Activation of the Sonic hedgehog signaling pathway, primarily through mutational inactivation of the PTCH1 gene, is associated with the development of basal cell carcinoma (BCC). Gli1, a member of the Gli family of transcription factors, is expressed in BCC and in transgenic mice targeted expression of Gli1 in basal keratinocytes leads to BCC development. In addition to BCC, previous studies have shown that Gli1 is expressed in the outer root sheath (ORS) of the hair follicle but is absent in interfollicular epidermis. In this study, we have characterized the expression pattern of two protein kinase C (PKC) isoforms expressed in BCC and hair follicles. We have then used reporter assays to investigate the effects of these isoforms on Gli1 transcriptional activity. We report that in BCC sections, PKCalpha but not PKCdelta was weakly expressed in the epidermis, whereas in the hair follicle, PKCalpha was expressed in the ORS and PKCdelta in the inner root sheath. In contrast, neither PKCalpha nor PKCdelta was expressed in BCC tumor islands, although both isoforms were often expressed in the surrounding stroma. In mammalian 293T cells, coexpression of constitutively active PKCalpha reduced the activity of Gli1 in a dose-dependent manner, whereas constitutively active PKCdelta increased the activity of Gli1, although this required higher expression levels. Regulation of mutant Gli1 protein localized exclusively to the nucleus was similar to that of the wild-type protein, indicating that nuclear-cytoplasmic shuttling is not a determinant of Gli1 control by either PKC isoform. Furthermore, PKC regulation of Gli1 did not involve activation of mitogen-activated protein kinase signaling. Finally, we show that exogenous Gli1 does not alter the expression of PKCalpha in human primary keratinocytes, suggesting that loss of this isoform in BCC is not via Hedgehog signaling. As BCCs have been proposed to originate from the ORS, loss of PKCalpha expression may be relevant to tumor formation; this may, in part, be because of the predicted increase in Gli1 transcriptional activity.

Basal Cell Carcinoma↗

PubMed1984

Distinct epitopes are recognized by cytolytic T lymphocyte clones on the same class I molecule: direct demonstration using DNA-transfected targets and long-term cytolytic T cell clones.

By producing long-term, stable, cytolytic T lymphocyte clones and utilizing targets expressing only a single gene product derived from the stimulator mouse strain, we have been able to directly demonstrate that T cells recognize distinct epitopes expressed on a single H-2 molecule. These multiple determinants are distinguishable by inhibition patterns with monoclonal antibodies (mAb). When two T cell clones, P-2.14 and P-2.17, are tested on an L cell transfected with the Dp gene (lambda 12a), the T cells kill the transfected targets as well as blasts derived from Dp mouse strains. mAb 7-16.10 inhibits recognition and killing of Dp targets by both P-2.14 and P-2.17. This mAb recognizes a private specificity H-2.m22. Interestingly mAb 11-20.3 which also recognizes the H-2.m22 specificity inhibits clone P-2.14 but not P-2.17. The mAb 7-16.10, however, competes with 11-20.3 for binding to the surface of L cells expressing the Dp gene. Thus the two T cells must recognize an overlapping specificity. Other mAb which bind to the H-2Dp molecule are unable to inhibit either of these two cytolytic T cell clones. Paradoxically, any of the mAb when tested individually are sufficient to inhibit the polyclonal response derived from in vitro mixed lymphocyte culture. Therefore, by using targets expressing only a single H-2 molecule derived by DNA-mediated gene transfer and cytolytic T cell clones we have been able to directly demonstrate the presence of multiple epitopes on a single molecule and define their inhibition with mAb directed to that same molecule.

Animals↗

PubMed1999

Transcriptional targeting of retroviral vectors to the erythroblastic progeny of transduced hematopoietic stem cells.

Targeted expression to specific tissues or cell lineages is a necessary feature of a gene therapy vector for many clinical applications, such as correction of hemoglobinopathies or thalassemias by transplantation of genetically modified hematopoietic stem cells. We developed retroviral vectors in which the constitutive viral enhancer in the U3 region of the 3' LTR is replaced by an autoregulatory enhancer of the erythroid-specific GATA-1 transcription factor gene. The replaced enhancer is propagated to the 5' LTR upon integration into the target cell genome. The modified vectors were used to transduce human hematopoietic cell lines, cord blood-derived CD34(+) stem/progenitor cells, and murine bone marrow repopulating stem cells. The expression of appropriate reporter genes (triangle upLNGFR, EGFP) was analyzed in the differentiated progeny of transduced stem cells in vitro, in liquid culture as well as in clonogenic assay, and in vivo, after bone marrow transplantation in lethally irradiated mice. The GATA-1 autoregulatory enhancer effectively restricts the expression of the LTR-driven proviral transcription unit to the erythroblastic progeny of both human progenitors and mouse-repopulating stem cells. Packaging of viral particles, integration into the target genome, and stability of the integrated provirus are not affected by the LTR modification. Enhancer replacement is therefore an effective strategy to target expression of a retroviral transgene to a specific progeny of transduced hematopoietic stem cells.

Animals↗

PubMed2002

Novel genomic cDNA hybrids produce effective RNA interference in adult Drosophila.

Drosophila melanogaster has been a premier genetic model system for nearly 100 years, yet lacks a simple method to disrupt gene expression. Here, we show genomic cDNA fusions predicted to form double-stranded RNA (dsRNA) following splicing, effectively silencing expression of target genes in adult transgenic animals. We targeted three Drosophila genes: lush, white, and dGq(alpha). In each case, target gene expression is dramatically reduced, and the white RNAi phenotype is indistinguishable from a deletion mutant. This technique efficiently targets genes expressed in neurons, a tissue refractory to RNAi in C. elegans. These results demonstrate a simple strategy to knock out gene function in specific cells in living adult Drosophila that can be applied to define the biological function of hundreds of orphan genes and open reading frames.

ATP-Binding Cassette Transporters↗

PubMed1999

Changes in microvessel endothelial cell gene expression in an in vitro human breast tumour endothelial cell model.

Selective targeting of tumour-endothelium has been proposed as a means of therapy. The successful exploitation of this approach will rely on the identification of suitable targets expressed specifically on the tumour-associated endothelium. In an attempt to identify novel tumour-endothelium associated targets we have used differential mRNA display to identify genes up-regulated in an in vitro breast tumour-endothelial cell culture model. Confluent monolayers of human mammary microvessel endothelial cells (HuMMEC) were incubated for 5 days with MDA-MB-231 breast adenocarcinoma cell-conditioned medium (TCM). mRNAs isolated from TCM-treated and control cells were amplified using 104 combinations of four 3(') anchored T(12)VN primers and 26 'random' 10mers by RT-PCR and the products examined on DNA sequencing gels. Seventy-four sequences were cloned and the differential expression of five genes was confirmed using dot-blots. These were identified as procollagen type-IV, Tie-2/Tek receptor tyrosine kinase, NADH dehydrogenase subunit-6, and ferritin heavy-chain, which were up-regulated, and insulin-like growth factor binding protein-5, which was down-regulated. Increased endothelial expression of basement membrane proteins and tyrosine kinase receptors is known to occur during angiogenesis. Our data support the use of this model for further in vitro investigation of tumour angiogenesis.

Journal Article↗