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At least 163 records · Page 9Linked to original sources
PubMed1990

Establishment of a pancreatic beta cell line that retains glucose-inducible insulin secretion: special reference to expression of glucose transporter isoforms.

Two cell lines have been established from insulinomas obtained by targeted expression of the simian virus 40 T antigen gene in transgenic mice. These cell lines, designated MIN6 and MIN7, produce insulin and T antigen and have morphological characteristics of pancreatic beta cells. MIN6 cells exhibit glucose-inducible insulin secretion comparable with cultured normal mouse islet cells, whereas MIN7 cells do not. Both cell lines produce liver-type glucose transporter (GT) mRNA at high level. Brain-type GT mRNA is also present at considerable level in MIN7 cells, but is barely detectable in MIN6 cells, suggesting that exclusive expression of the liver-type GT is related to glucose-inducible insulin secretion. MIN6 cells do not express either major histocompatibility (MHC) class I or class II antigens on the cell surface. However, treatment with interferon-gamma induces high levels of MHC class I antigens, and a combination of interferon-gamma and tumor necrosis factor-alpha induces a MHC class II antigen on the cell surface. These results emphasize that the MIN6 cell line retains physiological characteristics of normal beta cells. The MIN6 cell line will be especially useful to analyze the molecular mechanisms by which beta cells regulate insulin secretion in response to extracellular glucose concentrations. We discuss a possible role of GT isoforms in glucose sensing by beta cells.

Animals↗
PubMed2026

Antibody-drug conjugates in selected solid tumours: a position statement update based on findings from the third workshop held by the ETOP IBCSG Partners Foundation.

The European Thoracic Oncology Platform (ETOP) International Breast Cancer Study Group (IBCSG) Partners Foundation initiated a series of workshops for experts to review current evidence and offer recommendations to guide future antibody-drug conjugate (ADC) research. Here, we summarise key findings from the third workshop, which included experts in various solid tumours, basic/translational research scientists and pharmaceutical industry representatives. Recent positive phase III trial data have further incorporated ADCs into the standard of care [e.g. lung: sacituzumab tirumotecan; breast: trastuzumab deruxtecan (T-DXd), sacituzumab govitecan, datopotamab deruxtecan; muscle-invasive bladder cancer: enfortumab vedotin; ovarian cancer: mirvetuximab soravastine; cervical cancer: tisotumab vedotin]. Thus, research priorities must be tailored according to tumour type, potentially focussing initially on settings where ADCs could replace chemotherapy. Many phase III ADC trials have been initiated based on positive phase I data and although these trials are larger than those conducted historically, prespecified criteria (e.g. patient numbers and magnitude of efficacy) should be met to justify proceeding directly to phase III. Importantly, although several ADCs have been successfully developed without mandatory biomarker selection, biomarker-driven ADC development enables rational patient selection, as illustrated by multiple ADCs (e.g. T-DXd, mirvetuximab soravtansine and telisotuzumab vedotin). The identification, development and validation of predictive biomarkers are therefore essential, particularly given several critical nuances, including the algorithms used to assess biomarker status and the type of specimen analysed, all of which may be influenced by temporal and spatial heterogeneity. Additional ADC research priorities include the optimisation of ADC constructs to enhance efficacy/tolerability and the identification of reliable ADC targets, including work to elucidate attributes of already-identified targets. Finally, considering the vast amount of ADC-related data being generated, artificial intelligence could be leveraged to analyse combined datasets and generate composite biomarkers, including tumour histology, optimal target expression thresholds, molecular alterations and activated pathways affecting payload activity and target function, to accelerate research.

Humans↗
PubMed1996

Identification and characterization of the genes of Enterococcus faecalis plasmid pCF10 involved in replication and in negative control of pheromone-inducible conjugation.

The prgB gene of the Enterococcus faecalis pheromone-inducible conjugative plasmid pCF10 encodes the surface protein Asc10. This protein mediates cell aggregation and its expression results in high-frequency transfer of the plasmid from donor to recipient. To identify the minimum region necessary for negative regulation of prgB expression, target plasmids were constructed containing a recently identified positive control region and a prgB::lacZ transcriptional fusion; expression of prgB in cells carrying these plasmids was thus verified by beta-galactosidase assay. The target plasmids were used in genetic studies with compatible plasmids containing cloned pCF10 genes supplying putative negative control functions to define the minimum region of pCF10 required for shutdown of prgB expression in the absence of exogenous pheromone. The minimum segment required for negative control, as indentified by deletion analysis, was a 6.9-kb region extending from the 5' end of a gene called prgN, through a previously identified gene, prgX. The DNA in this region, which had not been previously characterized (2.85 kb), was sequenced, and several potential regulatory genes and plasmid replication genes were identified. Genetic analysis indicated that the prgN, -Y, and -X genes are involved in negative control; prgW may also play a role in negative control, since it appeared to be required for expression of prgY. prgX, or a closely adjacent DNA sequence, acted in cis. The region of pCF10 containing negative control genes was also shown to function as an autonomous replicon in E. faecalis.

Amino Acid Sequence↗
PubMedDate not supplied

Transgenic targeting of neuroendocrine peptide genes in the hypothalamic-pituitary axis.

A large number of neuroendocrine peptide genes have been tested for their ability to target expression to the hypothalamus and pituitary in transgenic mice. This has resulted in a number of powerful applications, for example, ablation or immortalization of specific cell types, and analysis of transcription regulatory sequences. The greatest amount of success in targeting cells of the neuroendocrine axis has been in the pituitary and has utilized regulatory sequences of genes that are normally expressed in pituitary. Greater difficulties have been encountered in directing expression to specific neurons in the hypothalamus. A primary goal of this review is to consider collectively the data obtained by a number of laboratories in order to draw conclusions about the general sequence requirements for achieving cell-specific expression. The data suggest that the mechanisms controlling cell-specific expression of neuropeptide genes in the hypothalamus are complex and involve multiple regulatory elements that may reside within the gene or many kilobases away from the promoter. These elements act positively and negatively in different cells to enhance or restrict expression, and may include sequences that shield a transgene from regulatory influences of other genes near the point of chromosomal insertion.

Animals↗
PubMed1996

Mucin-1-related T cell infiltration in colorectal carcinoma.

Mucins (MUC) are highly glycosylated molecules widely expressed on epithelia of different origins, including colonic mucosa. Altered glycosylation processes in tumour cells result in the exposure of normally cryptic peptide epitopes, which may then be recognized as tumour-specific antigens. Recently, MUC1-specific antibodies were detected in the serum of a broad range of cancer patients, and from different tumours tumour-specific cytotoxic T lymphocytes (CTL) were isolated that recognized MUC1. Absence of HLA restriction in the recognition has been ascribed to the highly repetitive sequence of the polypeptide core, allowing simultaneous recognition of multiple identical epitopes and cross-linking and aggregation of T cell receptor on mucin-specific T cells. We investigated the expression of MUC1 epitopes in 56 cell suspensions from Dukes' B to D colorectal carcinomas using antibodies that recognize distinct peptide sequences on the glycosylated or deglycosylated MUC1 protein backbone. No relation was observed between MUC1 expression, or the extent of its glycosylation, and Dukes' stage, tumour location and tumour differentiation, but a positive correlation was detected between the percentages of tumour cells expressing mucin-1 and the numbers of CD3+ infiltrating cells. These tumour-infiltrating lymphocytes contained, however, only a few MUC1-specific T lymphocytes, as CTL showing preferential killing of MUC1-expressing target cells were only obtained from one tumour. Since, in addition, the majority of colorectal carcinomas were found to express the fully glycosylated MUC1 glycoprotein, its potential role as a target antigen for T-lymphocyte-mediated immunotherapy in this tumour type is probably limited.

Antibodies, Monoclonal↗
PubMed1994

DNA regulatory sequences of the rat tyrosine hydroxylase gene direct correct catecholaminergic cell-type specificity of a human growth hormone reporter in the CNS of transgenic mice causing a dwarf phenotype.

Transgenic mice bearing 4.8 kilobases (kb) of upstream rat tyrosine hydroxylase (TH) sequences linked to a human growth hormone gene (hGH) exhibited cell-specific expression of hGH in all the appropriate catecholaminergic neurons in the central nervous system (CNS), although with different penetrance in two different mouse lineages. No ectopic expression was observed in any brain or peripheral region in one founder and its progeny. In another founder there was some ectopic expression in addition to appropriate and high levels of tissue-specific expression in all catecholaminergic areas. These results identify regulatory sequences that are sufficient for targeting expression to all catecholaminergic CNS neurons. Also, expression of exogenous hGH in the hypothalamus caused a dwarf phenotype, generating a novel genetic model for GH deficiency of hypothalamic origin.

Aging↗
PubMed1994

GFAP promoter directs astrocyte-specific expression in transgenic mice.

Glial fibrillary acidic protein (GFAP) is an intermediate-filament protein expressed abundantly and almost exclusively in astrocytes of the CNS. We are studying transcriptional regulation of the GFAP gene to gain insight into astrocyte function and also to develop an astrocyte-specific expression system for manipulating brain physiology. In this work, we have produced transgenic mice carrying the bacterial lacZ reporter gene linked to a 2.2 kilobase 5'-flanking sequence derived from the human GFAP gene that previously was shown to direct astrocyte-specific transcription in cultured cells. We report that this promoter directs expression to astrocytes in the CNS. In addition, the upregulation of GFAP gene activity that follows injury to the brain was mimicked by the transgene. One of the transgenes was found to be X-linked and appeared to undergo the usual random inactivation that achieves gene dosage compensation in females. The brains of hemizygous females stained uniformly rather than displaying mosaic patches, indicating that astrocytes intermingle following their formation. The specific expression of the GFAP-lacZ transgene means that it is now possible to target expression of other heterologous genes to astrocytes in vivo, and to study the mechanisms for reactive gliosis at the DNA level.

Animals↗
PubMed1995

Hyperpigmentation and melanocytic hyperplasia in transgenic mice expressing the human T24 Ha-ras gene regulated by a mouse tyrosinase promoter.

The tyrosinase promoter has been used to target expression of the mutated human T24 Ha-ras oncogene in pigment-producing cells of transgenic mice. Two independent founder mice carrying the transgene survived and showed the same distinct phenotype of mutated coat color, deeply pigmented skin with multiple nevi, and twirling behavior. The offspring of one of these founders were developed into a line that stably expressed the same phenotype. Histopathological analysis of the tissues revealed hyperpigmentation and/or melanocytic hyperplasia in the skin, eyes, inner ear, and meningeal membranes in the brain. Reverse transcriptase-polymerase chain reaction analysis revealed expression of the transgene in skin, brain, and spleen. We propose that these transgenic mice will be a model for studying the process of multistage melanoma carcinogenesis and a system for evaluating potential chemopreventive agents.

Animals↗
PubMed1995

Restricted tissue-specific but correct developmental expression mediated by a short human alpha 1AT promoter fragment in transgenic mice.

The tissue-specific and developmental pattern of expression controlled by the proximal promoter (position-348 to +15) derived from the human alpha-1-antitrypsin (h alpha 1AT) gene was studied in transgenic mice. The short promoter segment was linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The transgene showed highly specific expression in the liver and the correct developmental pattern of regulation. Interestingly, this short promoter targets expression to the liver with a greater specificity than that reported for larger alpha 1AT promoter fragments.

Animals↗
PubMed1991

Induction of HIV-specific CTL and antibody responses in mice using retroviral vector-transduced cells.

Recombinant retroviral vectors can efficiently transduce and express foreign genes in mammalian cells. We have examined the utility of retroviral vector-mediated gene transfer to deliver genes which encode human immunodeficiency virus type I (HIV) antigens capable of stimulating specific immune responses. Murine fibroblast cell lines were transduced with a nonreplicating murine retroviral vector carrying the gene encoding the HIV-IIIB envelope protein and were shown to express the gp160/120 protein. Mice immunized with syngeneic vector-transduced cells developed CD8+, class I major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) specific for targets expressing the HIV envelope protein. The CTL also exhibited lytic activity on target cells coated with synthetic peptides derived from the gp120 V3 hypervariable region of both the HIV-IIIB and HIV(MN) isolates. Following adoptive transfer in a murine tumor model, these CTL were shown to be effective in vivo by their ability to eliminate established tumor cells expressing the HIV protein. Vector-transduced syngeneic cells were also capable of eliciting HIV envelope-specific antibody responses in immunized mice. Sera obtained from these mice were found to bind to the HIV-IIIB gp160 protein as well as a peptide-defined neutralizing antibody epitope contained within the V3 domain of gp120. These sera exhibited virus-neutralizing activity in that they markedly reduced the ability of HIV to infect and form syncytia of a human T-cell line. This is the first demonstration that cells transduced with a retroviral vector encoding the HIV-IIIB envelope protein are capable of inducing effective HIV-specific cellular and humoral immune responses in mice.

Amino Acid Sequence↗
PubMed1992

Expression of diphtheria toxin A-chain in mature B-cells: a potential approach to therapy of B-lymphoid malignancy.

Specifically targeted expression of a toxin gene potentially represents a novel approach to cancer therapy. With a view to the ablation of B-cell malignancies, we have constructed a plasmid, designated pTHA71, which expresses the A-chain of diphtheria toxin (DT-A) with high efficiency and specificity in transfected, mature B-lymphoid cells. The construction incorporated immunoglobulin (Ig) kappa light chain gene regulatory sequences, including a kappa promoter, small intron, partial constant region exon, and 3'-flanking sequence (but lacking a known enhancer). These sequences conferred substantially more efficient expression of DT-A in mature B-cells than was seen from constructs that included only Ig promoters and enhancers. When transfected into the 70Z/3 murine pre-B-cell line, pTHA71 was only expressed efficiently if the cells were induced to express their endogenous, rearranged Ig kappa gene by prior exposure to lipopolysaccharide. The insertion of the enhancer from the Ig kappa large intron into pTHA71, generating pTHA81, did not markedly influence the level of DT-A expression in 70Z/3 cells. The observed absence of expression in pre-B-cells suggests that DT-A constructs similar to pTHA71 might be used for the therapeutic ablation of malignant B-cells of mature stages, while sparing normal progenitor cells.

Animals↗
PubMed1996

Altered epidermal cell growth control in vivo by inducible expression of transforming growth factor beta 1 in the skin of transgenic mice.

An inducible bovine KIV* keratin gene promoter was used to target expression of latent or activated transforming growth factor beta 1 (TGF beta 1) to keratinocytes in transgenic mice. This short (2.2-kb) keratin 6 (K6) promoter element was generally silent in untreated animals but was induced in keratinocytes when placed in culture or, in vivo, in response to hyperplasia that follows topical application of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All of the K6-TGF beta 1 transgenic lines studied showed attenuation of the basal keratinocyte proliferative response to 12-O-tetradecanoylphorbol-13-acetate as a consequence of inducible TGF beta 1 gene expression. One of the six lines studied showed constitutive transgene expression at low levels in the skin, and this line had a 2- to 3-fold increase in epidermal DNA labeling index over control mice. Although in vitro TGF beta 1 is known to be a potent negative regulator of epithelial cell proliferation, in vivo TGF beta 1 has complex biological activities and can act as either a positive or negative regulator of keratinocyte proliferation.

Animals↗
PubMed1983

Xenogeneic cytotoxic T-cell clones recognize alloantigenic determinants on HLA-A2.

Long-term murine cytotoxic T-cell clones arising in response to stimulation with human lymphoblastoid cells and reactive with the HLA-A2 antigen are characterized. These clones distinguish between HLA-A2 and 21 other serologically defined HLA-A and -B antigens. In addition, most clones discriminate between prototypical HLA-A2 antigens, expressed by the majority of HLA-A2-positive individuals, and variant HLA-A2 antigens, which are serologically identical with the prototype, but distinguishable by human cytotoxic T cells and by biochemical analysis. This discrimination is reflected as an inability to cause any significant lysis of variant HLA-A2-expressing target cells at effector-to-target ratios 10- to 100-fold greater than those giving 50% lysis of prototype HLA-A2-expressing cells. By screening a panel of serologically HLA-A2-positive cells, a new variant HLA-A2-expressing cell line has been defined. The recognition patterns of these xenogeneic clones are suggested to reflect recognition of alloantigenic polymorphic determinants. Based on the strong bias in the xenogeneic T-cell repertoire for such determinants, we propose a model for T-cell recognition of class I products of the major histocompatibility complex.

Animals↗
PubMed1995

Expression of HLA-C molecules confers target cell resistance to some non-major histocompatibility complex-restricted T cells in a manner analogous to allospecific natural killer cells.

Specific HLA molecules have recently been shown to confer target cell resistance to lysis by some CD3- natural killer (NK) cells. For certain NK clones, resistance is governed by two specificities (NK1 and NK2) that are associated with particular HLA-C alleles: in general, target cells expressing Cw1, Cw3, Cw7, or Cw8 are susceptible to NK1 but resistant to NK2 clones, whereas target cells expressing Cw2, Cw4, Cw5, or Cw6 are susceptible to NK2 and resistant to NK1 cells. These two clusters of HLA-C alleles are distinguished by a dimorphism in the alpha 1 helical region, localized at amino acid positions 77 and 80. In this report, we show that highly enriched CD3+/CD56- cytotoxic T cell sublines and CD3-/CD56+ NK sublines derived from the same donor have identical cytolytic specificities when tested against a panel of allogeneic LCL and various HLA-B and -C transfectant cell lines. The lysis pattern of the allogeneic cells appeared to be related to the NK2 specificity for both effector cells: LCL expressing HLA-Cw2, Cw4, Cw5, or Cw6 alleles were lysed, while LCL expressing HLA-Cw1, Cw3, or Cw7 molecules were resistant. Resistance to lysis could be conferred to susceptible target cells by transfection with a Cw*0702 gene, while expression of a Cw*0602 gene did not provide protection. Similar patterns of HLA-C-mediated resistance were also found with two polyclonal T cell lines generated from the peripheral blood lymphocytes of unrelated donors. Thus, major histocompatibility complex (MHC) molecules that induced resistance to particular NK cells also regulated target cell resistance to lysis by these non-MHC-restricted effector T cells. For both types of effector cells, direct binding to HLA-C molecules was necessary to achieve inhibition since preincubation with mAb specific for class I molecules destroyed the protection from lysis of HLA-Cw7 expressing target cells. mAbs specific for CD3 and CD8 molecules had no influence on lysis or inhibition of the NK-like T cells. Formation of MHC complexes with particular peptides did not appear to be essential to confer resistance, since a cell line with defective peptide transporter genes (TAP genes), when transfected with an appropriate HLA-C allele, was as resistant to lysis as HLA-C transfectant lines with normal TAP function. These results suggest that HLA-C molecules may deliver negative regulatory signals to some non-MHC-restricted T cells in a manner similar to that described previously for particular NK cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1993

MHC class I expression on tumor targets inhibits natural killer cell-mediated cytotoxicity without interfering with target recognition.

NK cell-mediated killing is inversely proportional to the amount of MHC class I expression on certain tumor targets. Two hypotheses have been proposed to explain why class I-bearing targets are more resistant to NK cell-mediated lysis: 1) the presence of class I prevents NK cell recognition of a triggering molecule on the target cell surface, or 2) class I recognition transmits a separate inhibitory signal to the NK cell. To differentiate between these potential mechanisms, we have used cloned human CD16+/CD3- NK cells, the class I-deficient cell line C1R, and C1R cells expressing high levels of transfected HLA class I gene products. If class I expression blocks NK cell recognition of the targets, then proximal cell signaling events such as phospholipase C-mediated hydrolysis of membrane phosphoinositides should be decreased in the NK cells interacting with the class I transfectants. However, we found that increases in the level of target cell class I expressions did not decrease phosphoinositide turnover or calcium signaling in NK cells. We also examined the effect of treating HLA-transfected C1R cells with mAb specific for the transfected MHC class I gene product. If class I expression has a negative regulatory influence on NK cell activation, then treating the targets with anti-HLA mAb should block the transmission of this negative signal. Consistent with this notion, addition of anti-HLA mAb (either whole Ig or F(ab')2 fragments) led to increased lysis of the class I-transfected targets. In contrast, addition of isotype-matched mAb specific for other cell surface markers did not alter sensitivity to lysis. All of these results suggest that MHC class I expression on target cells can initiate inhibitory signals in NK cells without blocking access to target structures.

Calcium↗
PubMed1992

Immortalized hypothalamic gonadotropin-releasing hormone neurons.

The neuroendocrine hypothalamus has been intensively studied using whole animals and tissue slices. However, it has been difficult to approach questions at the molecular and cellular level. By targeting expression of the oncogene product, simian virus 40 T antigen, in transgenic mice using the regulatory domain of the rat gonadotropin-releasing hormone (GnRH) gene, we have produced specific hypothalamic tumours. These tumours have been cultured to produce clonal cell lines (GT-1 cells) that express T antigen, GnRH and many other neuronal markers, but do not express other hypothalamic hormones. These immortal cell lines have a distinctive neuronal phenotype, process the GnRH peptide accurately and secrete GnRH in a pulsatile pattern. Thus, by targeting oncogenesis to a defined population of neurons using the regulatory region of a gene that is expressed late in differentiation of that cell lineage, we have succeeded in immortalizing hypothalamic GnRH neurons. The GT-1 cell lines are an excellent model for future molecular, cell biological, physiological and biochemical investigations into the mechanisms involved in regulation of GnRH and the characteristics of an isolated central nervous system neuron. Their derivation demonstrates the utility of targeting tumorigenesis to specific differentiated neurons of the central nervous system in transgenic mice.

Animals↗
PubMed1993

Carcinogen-treated skin allografts rejected by T lymphocytes specific for class I but not class II MHC antigens.

Treatment of skin with the chemical carcinogen 7,12-dimethylbenz[a]anthracene (DMBA), which reduces the density of epidermal class II MHC-expressing Langerhans cells (LC), enhances its survival when transplanted onto histoincompatible hosts. We have examined the ability of T lymphocytes which reject DMBA-treated skin to lyse P388D1 cells expressing either only class I or class I and II antigens. Lymphocytes isolated from solvent-treated grafts showed greater cytotoxicity for the targets expressing both antigens, indicating that some of these lymphocytes were specific for class II MHC antigens. In contrast, lymphocytes isolated from carcinogen-treated grafts lysed both targets similarly and hence did not contain any cells specific for class II MHC antigens. Anti-class I MHC antibody blocked cytotoxicity by both leukocyte populations to similar extents, but anti-class II MHC antibodies preferentially blocked T cells isolated from the solvent-treated grafts. There was no difference in the phenotype of the cytotoxic cells isolated from solvent- and carcinogen-treated grafts. Thus, whereas solvent-treated skin grafts are rejected by T cells specific for class I and II MHC antigens, DMBA-treated skin grafts are only rejected by class I MHC-specific T cells which may account for the enhanced survival of the carcinogen-treated grafts.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1995

In vivo viability of postmitotic Purkinje neurons requires pRb family member function.

The product of the retinoblastoma susceptibility gene, pRb, is known to be an important regulator of cell division. Disrupted central nervous system development in RB null mice suggests a critical function for pRb in the proliferative arrest and initiation of terminal differentiation of certain neurons. Previously, we have shown that SV40 T-ag expression targeted to Purkinje neurons in transgenic mice causes cell-specific death. Here we describe that T-ag expression induces DNA synthesis and results in DNA fragmentation in Purkinje neurons. Characterization of transgenic mouse lines expressing mutant T-ags demonstrate that the pRb binding domain of T-ag is required for induction of Purkinje cell loss. These findings indicate that a pRb function is required well beyond the completion of Purkinje neuron differentiation and provide a link between cell cycle regulation and neurodegeneration in vivo.

Animals↗