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PubMed1985

Natural killer cell recognition of target cells expressing different antigens of vesicular stomatitis virus.

Natural killer (NK) cells have the capability of lysing virus-infected, transformed, and embryonal cells, yet the nature of the target structure(s) recognized remains unclear. The availability of well-characterized temperature-sensitive (ts) mutants of vesicular stomatitis virus, defective in expression of individual viral-encoded polypeptides at the nonpermissive temperature (39 degrees C), offered an approach to elucidating NK-cell recognition of virus-infected cells. Target cells were infected with ts mutants in three functions: the viral surface glycoprotein (G protein; ts 045); the matrix (M) protein (ts G31, ts G33), and the polymerase (ts G11). Cells infected with wild-type virus and all ts mutants at the permissive temperature (31 degrees C) were killed by murine spleen cells. Similar to results on cytotoxic T lymphocytes, target cells infected by ts 045 defective in expression of G protein at 39 degrees C were not killed by NK cells. Unexpectedly, cells infected at 39 degrees C with the M-protein mutants also were not killed, although G protein was expressed at the cell surface. Target binding studies indicated that conjugates were not formed by cells infected with the ts mutants at the nonpermissive temperature. That expression of G protein was not sufficient for NK cell-mediated cytotoxicity was established in experiments in which a plasmid (pSVGL) containing the gene for vesicular stomatitis virus G protein was transfected into COS cells. Although G antigen was expressed on the plasma membrane, the cells were not lysed. These results suggest either that recognition of virus-infected cells depends on an appropriate conformation imparted to the viral G protein by association with the M protein or that NK cells can recognize alterations in the structure of the cell membrane induced by insertion of viral M and G molecules.

Animals↗
PubMed1997

Induced PAI-1 mRNA expression and targeted protein accumulation are early G1 events in serum-stimulated rat kidney cells.

Expression of plasminogen activator inhibitor type-1 (PAI-1), a member of the SERPIN gene family that functions to regulate the plasmin-based pericellular proteolytic cascade, is growth state-regulated in normal rat kidney (NRK) cells (Ryan and Higgins, 1990, J. Cell. Physiol., 155:376-384; Ryan et al., 1996, Biochem. J., 314:1041-1046). Comparative analysis of arrest states induced in NRK cells upon exposure to serum-deficient (0.5% FBS) or serum-free culture conditions served to define the kinetics of PAI-1 gene expression and fate of de novo-synthesized PAI-1 protein. While cells rendered quiescent in serum-free or serum-deficient media were equivalent with regard to the time course of PAI-1 mRNA induction, the level of expressed transcripts (27-fold vs. 12-fold) and accumulated saponin fraction PAI-1 protein (12-fold vs. 6-fold) were consistently greater in cells recruited into exponential growth phase from a serum-free as compared to a serum-deficient arrest condition. Relative PAI-1 mRNA abundance increased within 1-2 hr post-serum addition, was maximal at 4 hr, and declined rapidly thereafter; this time course of expression coupled with placement of entry into DNA synthetic phase at approximately 12 hr after stimulation indicates that PAI-1 induction is an early-to-mid G1 phase event. Induced PAI-1 protein was evident immunocytochemically within 2 hr of serum stimulation as a peripheral "rim" of accumulated protein restricted to the cellular ventral surface at the plane of the substrate. No PAI-1 was detected between individual cells suggesting that this protein may be targeted directly to the undersurface region. By 6 hr post-stimulation, the rim of PAI-1 deposition increased in intensity and broadened to occupy approximately 30 to 50% of the total undersurface area. Double-label immunocytochemistry indicated that accumulated PAI-1 was deposited in close proximity to, but not actually within, vinculin-containing focal contact structures. Potential functionality of induced PAI-1 expression to either the initiation or maintenance of the serum-stimulated phenotype was assessed using antibodies to PAI-1. The IgG fractions of two different antisera which neutralize the ability of PAI-1 to complex with and thereby inhibit the catalytic activity of urokinase plasminogen activator significantly reduced (by 25-35%) the incidence of cells displaying the serum-stimulated phenotype; antibodies that bind PAI-1 but do not block PAI-1 inhibitory activity were without effect. In view of the vagaries of antibody accessibility and in situ neutralizing activity (particularly in a region as structurally complex as the focal contact), these data may actually underestimate the importance of PAI-1 in maintaining the activated phenotype.

Animals↗
PubMed2000

T-cell killing of heterogenous tumor or viral targets with bispecific chimeric immune receptors.

We have previously described several novel chimeric immune receptors (CIRs) that redirect human T cells to kill malignant or HIV-infected cells. These CIRs comprise a cancer- or virus-specific ligand or single-chain antibody fused to the signaling domain of the T-cell receptor CD3-zeta subunit. Binding of the ligand- or antibody-based CIR to the target antigen (Ag) triggers T-cell-mediated cytolysis of the tumor- or virus-infected cell independent of target cell major histocompatibility complex class I expression. A new type of CIR was developed to mediate the lysis of cells that expressed one or more distinct viral or tumor Ags; three bispecific CIRs (BCIRs) were generated that recognized the carcinoembryonic Ag (CEA) and TAG-72 tumor Ags or, alternatively, distinct epitopes in the HIV envelope (HIVenv). T cells expressing the antitumoral Ag BCIR lysed both CEA- and TAG-72-expressing targets and did not kill Ag-negative targets or target cells expressing other members of the CEA family. Similarly, T cells expressing the anti-HIVenv BCIR lysed target cells expressing both the wild-type HIVenv and a mutant HIVenv that lacked the epitopes recognized by the monospecific CIRs. This approach permits the generation of T cells with a broader spectrum of activity capable of killing virus-infected cells and malignant cells and reduces the potential of progression of disease due to Ag loss variants.

Amino Acid Sequence↗
PubMed1990

Expression of a chimeric VIP gene is targeted to the intestine in transgenic mice.

We showed previously that a gene construction that consisted of 5.2 kb of 5' flanking sequence, the first exon, and part of the first intron of the human gene encoding vasoactive intestinal peptide (VIP) fused to the reporter gene chloramphenicol acetyltransferase (CAT) fully mimicked the diverse behavior of the endogenous VIP gene when transfected into subclones of the human neuroblastoma cell line SK-N-SH (Waschek et al., 1988). To determine if the same sequences were sufficient to target expression of a reporter to VIP-producing tissues in the mouse, we initiated a pilot study in which we generated four transgenic mice or mouse lines that contained the VIPCAT fusion gene. Detectable levels of CAT were found in the ileum of either founder or offspring of each of the transgenic mouse lines. In all other tissues tested, CAT activity was either below the level of detection or the transgene was not expressed, with the exception of one mouse in which ectopic expression in the cerebellum was observed. The results indicate that the VIP sequences utilized were sufficient to direct expression of the transgene to the intestine, but not necessarily to other sites of VIP expression. To investigate what specific DNA sequences might confer VIP expression in the intestine and other sites, we analyzed further the VIP gene in SK-N-SH subclones using VIP/luciferase fusion gene constructions. A 0.6 kb DNA fragment located between 4.0 kb and 4.6 kb upstream from the VIP transcriptional start site was found to impart a high level of expression in one subclone and an increased degree of phorbol ester induction in another. These and other data indicate that multiple transcriptional elements control VIP expression in neuroblastoma cells and are candidates as mediators of VIP gene expression in the intact animal.

Animals↗
PubMed2000

Induction of human Cdc37 in prostate cancer correlates with the ability of targeted Cdc37 expression to promote prostatic hyperplasia.

The Cdc37 gene encodes a 50 kDa protein which targets intrinsically unstable oncoprotein kinases such as Cdk4, Raf-1, and src to the molecular chaperone Hsp90. This activity is thought to play an important role in the establishment of signaling pathways controlling cell proliferation. The budding yeast Cdc37 homolog is required for cell division and mammalian Cdc37 is expressed in proliferative zones during embryonic development and in adult tissues, consistent with a positive role in proliferation. Here we report that human prostatic tumors, neoplasias and certain pre-malignant lesions display increased Cdc37 expression, suggesting an important and early role for Cdc37 in prostatic transformation. To test the consequences of increased Cdc37 levels, transgenic mice expressing Cdc37 in the prostate were generated. These mice displayed a wide range of growth-related abnormalities including prostatic epithelial cell hyperplasia and dysplasia. These data suggest that the expression of Cdc37 may promote inappropriate proliferation and may be an important early step in the development of human prostate cancer.

Animals↗
PubMed1996

A novel population of CD4+CD56+ myelin-reactive T cells lyses target cells expressing CD56/neural cell adhesion molecule.

CD56 is a member of the neural cell adhesion molecule family expressed on cells of the central nervous system and also on NK cells. Previous studies suggest the involvement of CD56 in effector-to-target cell conjugation mediated by NK cells. It was shown recently that CD56 is also expressed by subpopulations of CD8+ and CD4+ T cells. The present study describes the functional characteristics of CD4+CD56+ T cell lines established from blood of multiple sclerosis patients by stimulation with myelin basic protein (MBP). CD4+CD56+, MBP-specific T cell lines were able to lyse MBP-pulsed target cells in an HLA class II-restricted fashion. At the same time, they mediated MHC-unrestricted lysis of CD56+ target cells such as CD56+ lymphoid or glial tumor cells, but not of the typical NK target, K562. A number of experimental results including separation of CD4+CD56+ T cells into CD56 high and low expressing populations, cold target inhibition, as well as killing of CD56-transfected cells indicate that homotypic CD56 interactions are involved in the MHC-unrestricted lysis. CD56 interactions are not sufficient but are required for effector/target interaction. Our findings raise the possibility that CD4+CD56+ T cells sharing properties of both typical Ag-specific Th0-like T cells and NK cells might be involved in damage of tissues expressing CD56/neural cell adhesion molecule, such as the central nervous system. Thus, we provide evidence for a novel mechanism that could lead to organ-specific autoreactivity.

Biomarkers↗
PubMed1995

Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter.

To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the reporter gene luciferase and the murine preproendothelin-1 promoter. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9-kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per microgram of protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. The further demonstrate the targeting capability of this system, we overexpressed the lipid-peroxidating enzyme, human 15-lipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localization in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins.

Animals↗
PubMed1998

Expression of target genes by coinfection with replication-deficient viral vectors.

An in vivo transcription system was developed by coinfection of cells with replication-deficient viral vectors. Recombinant baculovirus (AcT7HCVLuc) and fowlpox virus (FPVT7HCVLuc) carrying a cDNA of the hepatitis C virus (HCV) minigene encoding the HCV 5' untranslated region (UTR), a luciferase gene and the 3' UTR, including the 98 nt extra sequence, under the control of the T7 promoter were constructed. The HCV minigene was synthesized in various cells by coinfection with one of these two viruses and recombinant baculovirus (AcCAT7) or adenovirus (AdexCAT7) expressing T7 RNA polymerase under the control of a mammalian promoter. Only a low level of luciferase expression was obtained in cells coinfected with AcT7HCVLuc and either AcCAT7 or AdexCAT7. In contrast, high-level luciferase expression was detected when the same cells were coinfected with FPVT7HCVLuc and either AcCAT7 or AdexCAT7. We further constructed a recombinant fowlpox virus with its HCV minigene extended to contain the whole HCV core protein region. Significantly high levels of expression of HCV core protein were detected in MT-2, COS7 and Vero cells by coinfection with the recombinant fowlpox virus and AdexCAT7. A coinfection system consisting of recombinant fowlpox virus and AdexCAT7 was established for high level of expression of a target gene in various cells.

Adenoviridae↗
PubMed2000

A carboxyl-terminal region important for the expression and targeting of the skeletal muscle dihydropyridine receptor.

We have used the yeast two-hybrid technique and expression of truncated/mutated dihydropyridine receptors (DHPRs) to investigate whether the carboxyl tail of the DHPR is involved in targeting to junctions between the sarcolemma and sarcoplasmic reticulum in skeletal muscle. The carboxyl tail was extremely reactive in yeast two-hybrid library screens, with the reactivity residing in amino acids 1621-1647 and abolished by a point mutation (V1642D). Dysgenic myotubes were injected with cDNA encoding green fluorescent protein fused to the amino terminus of DHPRs truncated after either residue 1620 (Delta1621-1873) or residue 1542 (Delta1543-1873) or of full-length DHPRs with the V1642D mutation (V1642D). For either Delta1621-1873 or V1642D, the restoration of excitation-contraction coupling was reduced approximately 40%, and the number of functional DHPRs in the sarcolemma was reduced approximately 30%, compared with the wild-type DHPR. The restoration of excitation-contraction coupling and surface expression was more drastically reduced (by approximately 90 and approximately 55%, respectively) for Delta1543-1873. Fluorescence microscopy revealed that Delta1621-1873 and V1642D were concentrated in a longitudinally restricted region near the injected nucleus, whereas wild-type DHPRs were present relatively uniformly along the length of a myotube. The intensity of fluorescence was greatly reduced for Delta1543-1873, indicating a low level of protein expression. Thus, residues 1543-1647 appear to play a role in the biosynthetic processing, transport, and/or anchoring of DHPRs, with residues 1543-1620 being particularly important for expression.

Animals↗
PubMed1999

Expression and targeting of the apoptosis inhibitor, survivin, in human melanoma.

The newly described apoptosis inhibitor survivin is expressed in many human cancers and appears to play a critical part in both apoptosis regulation and cell cycle progression. Its potential role in malignant melanoma is unknown. In a panel of 30 malignant melanomas, survivin was strongly expressed in all cases (15 of 15) of metastatic malignant melanomas and 13 of 15 cases of invasive malignant melanomas by immunohistochemistry. In invasive malignant melanomas, survivin was also expressed in the in-situ component of the lesion. Survivin expression was found in all cases (11 of 11) of nevi, but not in melanocytes in sections of normal skin. The apoptosis inhibitor bcl-2 was expressed in 26 of 30 cases, but generally at lower levels than that of infiltrating lymphocytes. The mitotic index, as assessed by MIB-1 staining, was consistently higher in metastatic than invasive malignant melanomas. Assessment of apoptotic index by in situ end-labeling revealed extremely low rates of apoptosis in most malignant melanomas. Survivin expression by western blotting was detected in four human metastatic malignant melanoma cell lines but not in cultured normal human melanocytes. Transfection of both YUSAC-2 and LOX malignant melanoma cells with green fluorescence protein-conjugated survivin anti-sense or green fluorescence protein-conjugated survivin dominant negative mutant (Cys84Ala) [corrected] resulted in increased apoptosis in the absence of other genotoxic stimuli. Two-color flow cytometry confirmed that YUSAC-2 cells transfected with survivin anti-sense expressed less endogenous survivin and exhibited an increased fraction of cells with sub-G1 DNA content. These data demonstrate that apoptosis inhibition by survivin may participate in the onset and progression of malignant melanomas, and suggest that therapeutic targeting of survivin may be beneficial in patients with recurrent or metastatic disease.

Adult↗
PubMed1999

Forced MyHCIIB expression following targeted genetic manipulation of conditionally immortalized muscle precursor cells.

The ability to carry out gene targeting in somatic stem cells while maintaining their stem cell characteristics would have important implications for gene therapy and for the analysis of gene function. Using mouse myoblasts, we have explored this possibility by attempting to alter the promoter of a myosin heavy chain gene (MyHCIIB) characteristic of physiologically "fast" muscle so as to force its unscheduled expression in physiologically "slow" muscle fibers. Conditionally immortalized muscle precursor cells were transfected with a gene targeting construct designed to replace the MyHCIIB promoter with that for the carbonic anhydrase III gene (CAIII), which is highly expressed in slow muscle. A potentially targeted clone was isolated and differentiated in culture to form myotubes which expressed MyHCIIB. Cells from the same clone were injected into both slow and fast muscle of host mice, where they contributed to fiber formation. In slow muscle, the fibers derived from this clone did not express MyHCIIB; this may reflect an instability of the targeted MyHCIIB locus and/or a failure of the hybrid promoter to function in slow fibers in vivo. Nonetheless, we have demonstrated that a "promoter knock-in" gene targeting procedure can be used to generate unique MyHCIIB-expressing myotubes in culture and that conditionally immortalized myoblasts can be subjected to extensive passaging and genetic manipulation without losing their ability to form fibers in culture and in vivo.

Animals↗
PubMed1992

Co-localized but target-unrelated expression of vasoactive intestinal polypeptide and galanin in rat dorsal root ganglion neurons after peripheral nerve crush injury.

Expression of vasoactive intestinal polypeptide (VIP) and galanin in dorsal root ganglion (DRG) neurons is known to be induced by peripheral nerve injury. We investigated (1) whether VIP and galanin were co-expressed by DRG neurons and (2) whether such neurons innervated specified peripheral targets (visceral, cutaneous or muscular). An antibody to the 200 kDa neurofilament subunit (NF200) was used as a marker for large type-A cells in the DRG. VIP and galanin were respectively observed in 22% and 67% of DRG neurons at the L5 spinal level after crushing of the sciatic nerve. Most VIP-containing neurons were small type-B cells (about 90%) and approximately 95% of VIP-containing neurons also showed galanin-like immunoreactivity. Galanin was expressed by both large type-A and small type-B cells. Immunocytochemistry combined with a retrograde tracer revealed that about 70-80% of the small type-B cells in each sensory division displayed VIP-like immunoreactivity, and that most of the tracer-labeled neurons also expressed galanin. These findings suggest that the expression VIP and/or galanin in response to peripheral nerve crush injury is a property common to visceral, cutaneous and muscular sensory neurons.

Animals↗
PubMed2001

Reduction of target gene expression by a modified U1 snRNA.

Although the primary function of U1 snRNA is to define the 5' donor site of an intron, it can also block the accumulation of a specific RNA transcript when it binds to a donor sequence within its terminal exon. This work was initiated to investigate if this property of U1 snRNA could be exploited as an effective method for inactivating any target gene. The initial 10-bp segment of U1 snRNA, which is complementary to the 5' donor sequence, was modified to recognize various target mRNAs (chloramphenicol acetyltransferase [CAT], beta-galactosidase, or green fluorescent protein [GFP]). Transient cotransfection of reporter genes and appropriate U1 antitarget vectors resulted in >90% reduction of transgene expression. Numerous sites within the CAT transcript were suitable for targeting. The inhibitory effect of the U1 antitarget vector is directly related to the hybrid formed between the U1 vector and target transcripts and is dependent on an intact 70,000-molecular-weight binding domain within the U1 gene. The effect is long lasting when the target (CAT or GFP) and U1 antitarget construct are inserted into fibroblasts by stable transfection. Clonal cell lines derived from stable transfection with a pOB4GFP target construct and subsequently stably transfected with the U1 anti-GFP construct were selected. The degree to which GFP fluorescence was inhibited by U1 anti-GFP in the various clonal cell lines was assessed by fluorescence-activated cell sorter analysis. RNA analysis demonstrated reduction of the GFP mRNA in the nuclear and cytoplasmic compartment and proper 3' cleavage of the GFP residual transcript. An RNase protection strategy demonstrated that the transfected U1 antitarget RNA level varied between 1 to 8% of the endogenous U1 snRNA level. U1 antitarget vectors were demonstrated to have potential as effective inhibitors of gene expression in intact cells.

3T3 Cells↗
PubMed1990

Generation of hen egg lysozyme-specific and major histocompatibility complex class I-restricted cytolytic T lymphocytes: recognition of cytosolic and secreted antigen expressed by transfected cells.

Syngeneic cells exogenously supplied with hen egg lysozyme (HEL) or endogenously synthesizing HEL were used as antigen-presenting cells to induce major histocompatibility complex class I-restricted cytotoxic T lymphocytes (CTL). Immunization of C57BL/6 mice followed by repeated stimulation of their splenocytes in vitro with trypsinized HEL peptides led to the generation of CTL lines specific for trypsinized HEL peptides and restricted by H-2K. Immunization of C3H mice with a mixture of soluble native HEL and irradiated syngeneic spleen cells followed by in vitro stimulation of immune spleen cells with soluble HEL could in a few cases result in HEL-specific CTL able to kill syngeneic transfectant L cells secreting HEL (HELs) or expressing cytosol-targeted HEL (HELc). The use of HELs or HELc transfectant L cells as in vivo and in vitro immunogens was a potent way for eliciting HEL-specific polyclonal CTL. These CTL and two CD8+ clones were found to be H-2K restricted and specific for the 1-17 N-terminal HEL peptide. In addition, the anti-HEL CTL could also exhibit a significant cross-reactivity against unsensitized and HEL-untransfected targets expressing the K restriction element. This cross-reactivity was likely due to recognition of unidentified HEL mimicking peptides (self-derived?) presented by the MHC class I (H-2K or H-2K) molecule used as the restriction element for the specific recognition of HEL. The CTL raised after immunization with HELs or HELc transfectant cells were found to recognize both the HELs and HELc transfectant cells even though HEL was not detected in the latter after a 2- or 5-min radiolabeling pulse. Recognition of both HELs and HELc transfectant cells by a given CTL clone suggests that HEL subjected to two separate processing pathways, each depending on the initial subcellular localization, can ensure the generation of similar MHC class I peptide complexes.

Animals↗
PubMed2001

Increased transgene expression by the mouse tyrosinase enhancer is restricted to neural crest-derived pigment cells.

In this study, we have addressed the impact of the mouse tyrosinase enhancer on regulated expression from the mouse tyrosinase promoter during embryonic development. Stable and transient transgenic experiments using the reporter gene lacZ reveal that (1) expression is detected in neural crest-derived melanoblasts from E11.5 onward, (2) the enhancer does not increase transgenic expression in optic cup-derived pigment cells of the retinal pigment epithelium (RPE), and (3) expression in the telencephalon is not any longer detected. The importance of the enhancer for expression in pigment cells of the eye was further investigated in adult mice using an attenuated diphtheria toxin A gene. This demonstrated that in presence of the enhancer the transgene expression is specifically targeted to neural crest-derived melanocytes of the choroid and not, or slightly, to the RPE. This suggests that tyrosinase is differentially regulated in the two pigment cell lineages, and that this promoter can be used to target expression preferentially to the neural crest-derived melanocyte lineage.

Animals↗
PubMed2000

Structure of the mouse glutamate decarboxylase 65 gene and its promoter: preferential expression of its promoter in the GABAergic neurons of transgenic mice.

GABA is synthesized by glutamate decarboxylase (GAD), which has two forms, GAD65 and GAD67. To elucidate the molecular mechanisms of mouse GAD65 (mGAD65) gene expression, we isolated and characterized the mGAD65 gene. The mGAD65 gene was found to be divided into 16 exons and spread over 75 kb. The sequence of the first exon and the 5'-flanking region indicated the presence of potential neuron-specific cis-regulatory elements. We used transgenic mice to examine the expression pattern conferred by a 9.2-kb promoter-proximal DNA fragment of the mGAD65 gene fused to the bacterial lacZ reporter gene. Transgenic mice showed high beta-galactosidase activity specifically in brain and testis. They also showed characteristic patterns of transgene expression in olfactory bulb, cerebellar cortex, and spinal cord, a similar expression pattern to that of endogenous mGAD65. However, no transgene expression was observed in the ventral thalamus or hypothalamus, in which high mGAD65 gene expression levels have been observed. These results suggest that the 9.2-kb DNA fragment of the mGAD65 gene is associated with its tissue-specific expression and its targeted expression in GABAergic neurons of specific brain regions but that additional regulatory elements are necessary to obtain fully correct expression.

Amino Acid Sequence↗
PubMed1994

Activation of proglucagon gene transcription by protein kinase-A in a novel mouse enteroendocrine cell line.

The gene encoding proglucagon is expressed predominantly in the pancreas and intestine. The physiological importance of glucagon secreted from the islets of Langerhans has engendered considerable interest in the molecular control of proglucagon gene transcription in the endocrine pancreas. In contrast, little is known about the molecular control of proglucagon gene expression in the intestine. The recent demonstration that glucagon-like peptide-1 (GLP-1) secreted from the intestine is a potent regulator of insulin secretion and glucose homeostasis has stimulated renewed interest in the factors that control GLP-1 synthesis in the intestinal L-cell. To develop a model for the analysis of intestinal proglucagon gene expression, we have targeted expression of a proglucagon gene-simian virus-40 large T-antigen fusion gene to enteroendocrine cells in transgenic mice. These mice develop intestinal tumors that were used to derive a novel cell line, designated GLUTag, that expresses the proglucagon gene and secretes immunoreactive GLP-1 in vitro. GLUTag cells demonstrate morphological characteristics of enteroendocrine cells by electron microscopy and are plurihormonal, as shown by immunocytochemistry and RNA analyses. GLUTag cells express the proglucagon and cholecystokinin genes, consistent with the pattern of lineage-specific enteroendocrine differentiation described for mouse intestine. Proglucagon gene expression was induced by activators of the protein kinase-A pathway, and a combination of messenger RNA half-life and nuclear run-on experiments demonstrated that the protein kinase-A-induction is mediated by an increase in proglucagon gene transcription. In contrast, activators of protein kinase-C stimulated secretion, but not biosynthesis of the PGDPs in GLUTag cell cultures. Analysis of proglucagon processing in GLUTag cells demonstrated the liberation of glucagon, oxyntomodulin, glicentin, and multiple forms of GLP-1. These observations provide evidence for the direct induction of proglucagon gene transcription by a cAMP-dependent pathway and suggest that the GLUTag cell line represents a useful model for the analysis of the molecular determinants of enteroendocrine gene expression.

1-Methyl-3-isobutylxanthine↗
PubMed1994

Transformation-associated 86 kDa natural killer target molecule expressed on the mouse, rat and human cell surface.

We previously reported on the 86 kDa natural killer target molecule associated with transformation of the oncogene-transfected rat fibroblasts. This molecule may participate in the lethal hit phase of cytotoxicity by natural killer (NK) cells. Originally, this molecule was defined by mAb109, but mAb109 could react only with rat tumor lines. In this report, to determine whether the 86 kDa molecule could be utilized as a natural killer target molecule in mammalian cells, we developed a polyclonal anti-86 kDa antibody (pAb109). Our data indicated that pAb109 preferentially reacted with NK-susceptible lines such as mouse YAC-1, rat W31 and human K562 cells, but reacted only weakly with NK-resistant mouse EL-4, rat fetal fibroblast WFB and human fetal fibroblast HEPM. In a cytotoxicity experiment, pAb109 F(ab')2 fragments could inhibit the cytolysis by NK cells of W31 and K562 cells. However, these fragments did not inhibit the cytotoxicity of non-NK cells such as CD3+, CD4-, CD8- T cell receptor alpha beta- T cells (presumably gamma delta T cells) to W31 cells. Taken together, these data suggest that the cell transformation-associated 86 kDa molecule may be critical in NK cytotoxicity, and a candidate for the NK target structure in mammalian tumor cells.

3T3 Cells↗