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At least 109 records · Page 6Linked to original sources
PubMed2001

Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells.

Designing effective strategies to load human dendritic cells (DCs) with tumor antigens is a challenging approach for DC-based tumor vaccines. Here, a cytoplasmic expression system based on mRNA electroporation to efficiently introduce tumor antigens into DCs is described. Preliminary experiments in K562 cells using an enhanced green fluorescent protein (EGFP) reporter gene revealed that mRNA electroporation as compared with plasmid DNA electroporation showed a markedly improved transfection efficiency (89% versus 40% EGFP(+) cells, respectively) and induced a strikingly lower cell toxicity (15% death rate with mRNA versus 51% with plasmid DNA). Next, mRNA electroporation was applied for nonviral transfection of different types of human DCs, including monocyte-derived DCs (Mo-DCs), CD34(+) progenitor-derived DCs (34-DCs) and Langerhans cells (34-LCs). High-level transgene expression by mRNA electroporation was obtained in more than 50% of all DC types. mRNA-electroporated DCs retained their phenotype and maturational potential. Importantly, DCs electroporated with mRNA-encoding Melan-A strongly activated a Melan-A-specific cytotoxic T lymphocyte (CTL) clone in an HLA-restricted manner and were superior to mRNA-lipofected or -pulsed DCs. Optimal stimulation of the CTL occurred when Mo-DCs underwent maturation following mRNA transfection. Strikingly, a nonspecific stimulation of CTL was observed when DCs were transfected with plasmid DNA. The data clearly demonstrate that Mo-DCs electroporated with mRNA efficiently present functional antigenic peptides to cytotoxic T cells. Therefore, electroporation of mRNA-encoding tumor antigens is a powerful technique to charge human dendritic cells with tumor antigens and could serve applications in future DC-based tumor vaccines.

Antigen Presentation↗
PubMed1998

ERCC1 mRNA levels complement thymidylate synthase mRNA levels in predicting response and survival for gastric cancer patients receiving combination cisplatin and fluorouracil chemotherapy.

PURPOSE: We have previously shown that relative thymidylate synthase (TS) mRNA levels in primary gastric adenocarcinomas treated with fluorouracil (5-FU) and cisplatin are inversely associated with response and survival. This is a presumed function of TS as a target for 5-FU activity. We now test the hypotheses that the relative mRNA level of the excision repair cross-complementing (ERCC1) gene is inversely associated with response and survival as an independent function of cisplatin efficacy. PATIENTS AND METHODS: Patients had intact, untreated, primary gastric adenocarcinoma cancer and were evaluated for eligibility on a preoperative cisplatin infusion-5-FU protocol. cDNA, derived from primary gastric tumors before chemotherapy, was used to determine ERCC1 mRNA levels, expressed as the ratio of polymerase chain reaction (PCR) product of the ERCC1 gene and the beta-actin gene. RESULTS: The median ERCC1 mRNA level from 38 primary gastric cancers (33 assessable for response) was 5.8 x 10(-3) (range, 1.8 x 10(-3) to 19.5 x 10(-3)). Of 17 responding patients, 13 (76%) were less than or equal to 5.8 x 10(-3) and four were greater than 5.8 x 10(-3) (P = .003). The median survival for patients with ERCC1 mRNA levels less than or equal to 5.8 x 10(-3) has not been reached, whereas for those greater than 5.8 x 10(-3) it was 5.4 months (P = .034). The median TS mRNA level, 3.7 x 10(-3) (range, 0.9 to 18.9) also segregated responsive versus resistant tumors (P = .024). With both ERCC1 and TS mRNA levels below their medians, 11 of 13 patients (85%) responded; with both ERCC1 and TS mRNA levels above their medians, two of 10 patients (20%) responded (P = .003). CONCLUSION: Considered separately, either ERCC1 or TS mRNA levels in a primary gastric adenocarcinoma has a statistically significant relationship to response. ERCC1 mRNA levels have a statistically significant association with survival; in this cohort TS mRNA levels did not reach statistically significant association with survival as in our previous publication. Whether these molecular parameters are independent of each other as predictors of outcome remains to be determined.

Adenocarcinoma↗
PubMed2003

Parathyroid hormone (PTH) secretion, PTH mRNA and calcium-sensing receptor mRNA expression in equine parathyroid cells, and effects of interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha on equine parathyroid cell function.

Parathyroid hormone (PTH) is secreted by the chief cells of the parathyroid gland in response to changes in ionized calcium (Ca(2+)) concentrations. In this study, we measured PTH secretion, and PTH mRNA and calcium-sensing receptor (CaR) mRNA expression by equine parathyroid chief cells in vitro. We also evaluated the effects of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha on PTH secretion, and PTH and CaR mRNA expression. The relationship between PTH and Ca(2+) was inversely related. PTH secretion decreased from 100% (day 0) to 13% (day 30). PTH mRNA expression declined from 100% (day 0) to 25% (day 30). CaR mRNA decreased from 100% (day 0) to 16% (day 30). Chief cells exposed to high (2.0 mM) Ca(2+) concentrations had a lower PTH mRNA expression compared with low Ca(2+) concentrations. Ca(2+) concentrations had no effect on CaR mRNA expression. The inhibitory effect of high Ca(2+) concentrations on PTH secretion also declined over time. After day 10, there was no significant difference in PTH secretion between low and high Ca(2+ )concentrations. IL-1beta decreased both PTH secretion (75%) and PTH mRNA expression (73%), and resulted in a significant overexpression of CaR mRNA (up to 142%). The effects of IL-1beta were blocked by an IL-1 receptor antagonist. IL-1beta decreased the Ca(2+) set-point from 1.4 mM to 1.2 mM. IL-6 decreased PTH secretion (74%), but had no effect on PTH and CaR mRNA expression. TNF-alpha had no effect on PTH secretion, and PTH and CaR mRNA expression. In summary, the decreased responsiveness of parathyroid cells to Ca(2+) from 0 to 30 days can be explained, in part, by the reduced CaR expression. IL-1beta and IL-6 but not TNF-alpha affected parathyroid function in vitro and may be important in influencing PTH secretion in the septic horse.

Animals↗
PubMed1999

Downregulation of uncoupling protein 2 mRNA in white adipose tissue and uncoupling protein 3 mRNA in skeletal muscle during the early stages of leptin treatment.

The mechanisms underlying the increase in energy expenditure during leptin treatment are not clear. We recently showed that a 5-h intravenous or intracerebroventricular infusion of leptin elevated basal glucose uptake in skeletal muscle (SM) and brown adipose tissue and increased whole-body glucose turnover in C57Bl/6J mice (Kamohara S, Burcelin R, Halaas JL, Friedman JM, Charron MJ: Acute stimulation of glucose metabolism in mice by leptin treatment. Nature 389:374-377, 1997). We extended the previous study by measuring steady-state levels of uncoupling protein (UCP)-2 mRNA and UCP-3 mRNA in white adipose tissue (WAT) and SM. Leptin by intravenous or intracerebroventricular infusion for 5 h was associated with a decrease in UCP-2 mRNA in WAT (47-52%) and UCP-3 mRNA in SM (33-37%). Because overexpression of UCP-2 or UCP-3 can depolarize the inner mitochondrial membrane, suppression of UCP-2 mRNA and UCP-3 mRNA may in fact lower respiratory demands in WAT and SM. This is consistent with the parallel suppression of cytochrome oxidase subunit IV (COX-IV) mRNA in WAT (35-39%) after leptin infusion. COX-IV mRNA in SM did not respond to acute leptin treatment. Mitochondrial inorganic phosphate carrier (P1C) mRNA was also suppressed in WAT (33-35%) by either method of leptin infusion, but only intravenous infusion of leptin reduced P1C mRNA in SM (40%). Denervation suppressed mRNA levels for UCP-2 (49%), UCP-3 (36%), and COX-IV (59%) and eliminated the acute response to leptin in SM. The comparable response to leptin under intravenous or intracerebroventricular infusion and the loss of responsiveness after denervation strongly suggest that the acute effects of leptin involve central signaling pathways.

Adipose Tissue↗
PubMed1992

Colocalization of NGF binding sites, trk mRNA, and low-affinity NGF receptor mRNA in primary sensory neurons: responses to injury and infusion of NGF.

The distributions of mRNAs for the protooncogene trk and the low-affinity NGF receptor (LNGFR) were studied by hybridization with oligonucleotide probes on sections of adult rat primary sensory and sympathetic ganglia. For comparison with high-affinity binding sites, adjacent sections were processed for NGF receptor radioautography. Among neurons in lumbar dorsal root ganglia and trigeminal ganglia, trk mRNA and NGF-binding sites were closely colocalized; this finding together with previous direct evidence in other cell types is taken to indicate that trk protein is an essential component of the high-affinity NGF receptor in adult sensory neurons. In lumbar dorsal root ganglia and trigeminal ganglia, abundant LNGFR mRNA was found in all neurons with strong 125I-NGF labeling and on additional neurons lacking high-affinity NGF-binding sites. The presence of abundant LNGFR in neurons with high-affinity receptors could be the cause and/or consequence of their ability to respond to NGF. Neurons with abundant LNGFR mRNA but few high-affinity NGF-binding sites may have receptors for other members of the neurotrophin family. In nodose ganglia, neurons with high concentrations of LNGFR mRNA greatly outnumbered the small percentage with abundant trk mRNA. Following intrathecal infusion of NGF to otherwise normal dorsal root ganglia, the concentrations of LNGFR mRNA but not those of trk mRNA and NGF-binding sites were increased in NGF-responsive neurons. The usual single normal pattern of frequency histograms of LNGFR labeling indices became bimodal in response to NGF. Concentrations of NGF-binding sites, LNGFR mRNA, and trk mRNA were all decreased by peripheral nerve transection and restored by exogenous NGF, the restoration being complete for LNGFR mRNA and partial for trk mRNA and NGF-binding sites. The data indicate that NGF can regulate both LNGFR and trk mRNAs but do not clarify the possible contribution of the LNGFR protein to high-affinity binding sites.

Amino Acid Sequence↗
PubMed2004

[Detection of alpha-fetoproteins mRNA and melanoma antigen-1 mRNA in peripheral blood of patients with hepatocellular carcinoma and its clinical significance].

OBJECTIVE: To explore the feasibility of alpha-fetoproteins (alpha-FP) mRNA and melanoma antigen-1 (MAGE-1) mRNA as 2 markers of micrometastasis in hepatocellular carcinoma (HCC) patients' peripheral blood. METHODS: Subjects were divided into five groups: patients with HCC (n=65); patients with hepatitisB and liver cirrhosis (n=22); patients with metastatic liver cancer (n=12); patients with hepatic hemangioma (n=12); and healthy volunteers (n=20). alpha-FP mRNA and MAGE-1 mRNA in peripheral blood were tested by nested reverse transcription polymerase chain reaction (RT-PCR). RESULTS: MAGE-1 mRNA and alpha-FP mRNA were detected in 27 (42%) and 35 (54%) of 65 HCC patients respectively, while 42 (65%) HCC patients were positive for at least 1 marker. The frequency of alpha-FP mRNA and MAGE-1 mRNA positivity strongly correlated with portal thrombosis, intrahepatic metastatic nodules, tumor diameter and TNM stage (P <0.05). With regard to controls, 4 of 12 (33%) samples from patients with metastatic liver cancer showed MAGE-1 mRNA in their peripheral blood, but groups of hepatitis and cirrhosis, hepatic hemangioma, healthy volunteers did not. And 3 of 22 samples (14%) from group hepatitis and cirrhosis showed alpha-FP mRNA, but none of samples from other control group showed alpha-FP mRNA. CONCLUSIONS: Nested RT-PCR is a sensitive and reliable method for detecting circulating HCC cells. Two-marker RT-PCR assay with a liver-specific alpha-FP marker in combination with a cancer-specific MAGE-1 marker may be a promising tool for detecting micrometastases with better sensitivity and specificity than one marker RT-PCR.

Adult↗
PubMed1989

5-Fluorouracil inhibits dihydrofolate reductase precursor mRNA processing and/or nuclear mRNA stability in methotrexate-resistant KB cells.

This laboratory previously reported that 5-fluorouracil (FUra) increases dihydrofolate reductase (DHFR) precursor mRNA (pre-mRNA) levels relative to DHFR mRNA levels in a methotrexate-resistant KB cell line; these data suggested that incorporation of FUra into RNA may, in part, lead to cell death through the inhibition of mRNA processing (Will, C. L., and Dolnick, B.J. (1987) J. Biol. Chem. 262, 5433-5436). Utilizing a methotrexate-resistant KB cell line designated 1BT, we now report the kinetic basis for altered levels of DHFR RNA observed in FUra-treated cells. Long-term exposure to FUra had no effect on the steady-state level of DHFR pre-mRNA containing intron V or I. However, steady-state levels of total DHFR mRNA decreased 2.0-fold on a per cell basis in cells exposed to 1.0 microM FUra. No significant change in the half-life of total DHFR mRNA or pre-mRNA was observed in cells exposed to FUra (t1/2 = approximately 11.5 h and 50 min, respectively). Nuclear/cytoplasmic RNA labeling experiments demonstrated that the rate of nuclear DHFR RNA conversion to cytoplasmic DHFR mRNA decreased approximately 1.8-fold in FUra-treated cells. These results provide further evidence the FUra may inhibit processing of mRNA precursors and/or affect the stability of nuclear DHFR mRNA.

Blotting, Northern↗
PubMed1985

Regulation of growth hormone mRNA synthesis by 3,5,3'-triiodo-L-thyronine in cultured growth hormone-producing rat pituitary tumor cells (GC cells). Dissociation between nuclear iodothyronine receptor concentration and growth hormone mRNA synthesis during the deoxyribonucleic acid synthesis phase of the cell cycle.

3,5,3'-Triiodo-L-thyronine (T3) regulates the growth rate and GH production of cultured GC cells, a rat pituitary tumor cell line. We have previously demonstrated a parallel increase in cellular content of DNA and nuclear T3 and glucocorticoid receptors during the DNA synthesis (S) phase of the GC cell growth cycle. To determine the relationship between the increase in nuclear hormone receptors and GH production in S-phase cultures, we measured the synthesis rate of GH by pulse-labeling with [3H]leucine and immunoprecipitation as well as the relative concentration of GH mRNA by dot hybridization employing formaldehyde-treated cytoplasm and GH cDNA. Total protein synthesis was similar in S-phase and asynchronous cultures. However, in comparison to asynchronous cultures, S-phase cells had an increased GH synthesis rate, p less than 0.005 (from 13,430 +/- 609 to 19,150 +/- 1160 cpm/10(6) cells/2 h) and increased GH mRNA, p less than 0.001 (from 7.2 +/- 1.2 to 14.5 +/- 1.5 relative A units). The S-phase-associated augmentation in GH production did not appear to result from a decrease in ADP-ribosylation induced by 2 mM thymidine treatment which was utilized for the S-phase synchronization. To determine whether increased GH mRNA and GH synthesis in S-phase was associated with an increase in synthesis of GH mRNA, we measured the incorporation of [3H]uridine into GH mRNA by incubating partially synchronized S-phase cells with [3H]uridine and isolating 3H-labeled GH mRNA by hybridization to GH cDNA immobilized on nitrocellulose filters. Total RNA synthesis was similar in asynchronous, S-phase and G1 cell populations. However, the mean incorporation of [3H]uridine into GH mRNA of S-phase cultures was decreased to 52, 59, and 61% (counts/min of GH mRNA/10(6) cells), 49, 59, and 65% (ppm of total RNA), and 64 and 69% (ppm of poly(A)+ RNA) of asynchronous cultures. Our studies show further that the decrease in [3H]uridine incorporation into GH mRNA did not result from a cell cycle specific change in efficiency of hybridization or exclusively to an S-phase associated increased rate of degradation of GH mRNA. Thus, despite increased nuclear T3 and glucocorticoid receptors and, increased GH mRNA and GH synthesis, the synthesis rate of GH mRNA appears decreased in S-phase GC cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1995

[Expression of endothelin-1 mRNA, endothelin receptor-A and nitric oxide synthase mRNA in pulmonary artery and right ventriculus cordis of rats exposed to hypoxia].

Endothelins (ETs) are a family of novel regulatory peptides which can constrict the vascular and promote the proliferation of vascular smooth muscle cells. Serum ET-1 was elevated, and nitric oxide (NO) levels were reduced in hypoxic pulmonary hypertension. However, the exact mechanism remains unclear, and no study has elucidated if hypoxia could stimulate directly overgrowth of right ventricle in patients with chronic cor pulmonale. To investigate the role of ET-1 and NO in hypoxia-induced pulmonary hypertension and right ventricular hypertrophy, we measured the levels of ET-1 mRNA, ET receptor-A (ETR-A) mRNA and nitric oxide synthase (NOS) mRNA in the pulmonary artery and right ventricle of rats exposed to hypoxia (FiO = 0.1, 8 hours daily for 1, 2 and 3 weeks) by reverse transcription-polymerase chain reaction (RT-PCR). ET-1 mRNA level of pulmonary artery raised after 1 week's hypoxia (P < 0.05), and after 2 weeks' hypoxia, it returned to near normal, but elevated significantly again after 3 weeks' hypoxia. Pulmonary artery ETR-A mRNA in 1 and 2 weeks hypoxic groups showed no significant change, but it raised significantly in 3 weeks' hypoxic group. After exposure to hypoxia for 1, 2 and 3 weeks, NOS mRNA in the pulmonary artery all reduced significantly (P < 0.05). The right ventriculus cordis showed a significant increase in weight after 3 and 2 weeks' hypoxia. ET-1 mRNA showed no significant change but ETR-A mRNA increased significantly after 2 weeks' hypoxia; both ET-1 mRNA and ETR-A mRNA showed significant increase in weight after 3 and 2 weeks' hypoxia. In the ventriculus cordis of rats exposed to hypoxia for 2 and 3 weeks, NOS mRNA had no significant change.

Animals↗
PubMed1977

Comparison of mRNA binding by Met-tRNAf binding protein and mRNA-associated proteins.

One of the heterogeneous mRNA binding activities in the 0.5 M KCl eluate of rabbit reticulocyte polyribosomes co-purified to apparent homogeneity through phosphocellulose and DEAE-cellulose chromatography and isoelectric focusing with the GTP-dependent Met-tRNAf binding protein. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following iodination revealed putative subunits of 51,000 and 39,000 apparent molecular weights. Specificity of mRNA binding by this protein was suggested since the ability of poly(A)-rich mRNA to compete for binding of [3H]poly(A)-rich mRNA exceeded by 10- to 100-fold that of most natural or synthetic RNAs tested, except for the hybrid poly(G) - poly(C) which was almost as effective, and poly(G), which was more effective, at competing for protein-dependent binding. The mRNA binding activity exhibited complete GTP independence and no apparent divalent cation requirement. GDP inhibited Met-tRNAf binding but neither GDP, GMP, nor 7-methylguanosine 5'-monophosphate inhibited mRNA binding by this protein. Similar data were obtained with respect to the ability of natural or synthetic RNAs to compete for binding of [3H]poly(A)-rich mRNA by proteins associated with purified rabbit reticulocyte polyribosomal mRNA-protein particles; while poly(A) was an ineffective competitor, poly(G) was more effective than even mRNA at competing for protein-dependent binding. No significant binding of Met-tRNAf by mRNA-protein particles was detected. Polyacrylamide gel electrophoresis following reduction of mRNA-protein particles revealed apparent co-migration of a major protein with one subunit of the GTP-dependent Met-tRNAf binding protein, but no protein comparable to the 39,000 dalton subunit protein.

Animals↗
PubMed2000

Cloning and characterization of mRNA capping enzyme and mRNA (Guanine-7-)-methyltransferase cDNAs from Xenopus laevis.

The mRNA cap structure, which is synthesized by a series of reactions catalyzed by capping enzyme, mRNA (guanine-7-)-methyltransferase, and mRNA (ribose-2'-O-)-methyltransferase, has crucial roles for RNA processing and translation. Methylation of the cap structure is also implicated in polyadenylation-mediated translational activation during Xenopus oocyte maturation. Here we isolated two Xenopus laevis cDNAs, xCAP1a and xCAP1b, for mRNA capping enzyme and one cDNA for mRNA (guanine-7-)-methyltransferase, xCMT1, which encode 598, 511, and 402 amino acids, respectively. The deduced amino acid sequence of xCAP1a was highly homologous to that of human capping enzyme hCAP1a, having all the characteristic regions including N-terminal RNA 5'-triphosphatase as well as C-terminal mRNA guanylyltransferase domains which are conserved among animal mRNA guanylyltransferases, whereas in xCAP1b the most C-terminal motif was missing. The amino acid sequence of xCMT1 was also similar to human (guanine-7-)-methyltransferase, hCMT1a, with all the conserved motifs among cellular (guanine-7-)-methyltransferases, except for its N-terminal portion. The recombinant xCAP1a and xCMT1 exhibited cap formation and mRNA (guanine-7-)-methyltransferase activities, respectively. RT-PCR analysis showed that mRNA for xCAP1a and xCMT1 exist abundantly in fertilized eggs as maternal mRNAs, but xCMT1 mRNA gradually decreased in its amount in later stages of early development.

Amino Acid Sequence↗
PubMed1995

The polyribosomal protein bound to the 3' end of histone mRNA can function in histone pre-mRNA processing.

Histone mRNAs end in a conserved 26 nt sequence which can form a stem-loop with a six-base stem and a four base loop. The 3' end of histone mRNA functions in the nucleus in pre-mRNA processing and mRNA transport and in the cytoplasm in translation and regulation of histone mRNA stability. The stem-loop binding protein (SLBP), found in both the polyribosomes and the nucleus, binds to the 3' end of histone mRNA. A nuclear extract which efficiently processes histone pre-mRNA has been prepared from mouse myeloma cells. The factors which bind the 3' end of histone mRNA can be depleted from this extract using a biotinylated oligonucleotide. Using the depleted extract, we show that the SLBP found in the polyribosomes can function in histone pre-mRNA processing, suggesting that the SLBP associates with histone pre-mRNA in the nucleus and accompanies the mature mRNA to the cytoplasm.

Animals↗
PubMed1994

Influenza virus NS1 protein inhibits pre-mRNA splicing and blocks mRNA nucleocytoplasmic transport.

The influenza virus RNA segment 8 encodes two proteins, NS1 and NS2, by differential splicing. The collinear transcript acts as mRNA for NS1 protein, while the spliced mRNA encodes NS2 protein. The splicing of NS1 mRNA was studied in cells transfected with a recombinant plasmid that has the cDNA of RNA segment 8 cloned under the SV40 late promoter and polyadenylation signals. As described for influenza virus-infected cells, NS1 mRNA was poorly spliced to yield NS2 mRNA. However, inactivation of the NS1 gene, but not the NS2 gene, led to a substantial increase in the splicing efficiency, as shown by the relative accumulations of NS1 and NS2 mRNAs. This effect was not specific for NS1 mRNA, since the splicing of the endogenous SV40 early transcript was altered in such a way that t-Ag mRNA was almost eliminated. These changes in the splicing pattern coincided with a strong inhibition of the mRNA nucleocytoplasmic transport. Both NS1 and NS2 mRNAs were retained in the nucleus of cells expressing NS1 protein, but no effect was observed when only NS2 protein was expressed. Furthermore, other mRNAs tested, such as T-Ag mRNA and the non-spliceable nucleoprotein transcript, were also retained in the nucleus upon expression of NS1 protein, suggesting that it induced a generalized block of mRNA export from the nucleus.

Amino Acid Sequence↗
PubMed2005

Double-stranded secondary structures on mRNA induce type I interferon (IFN alpha/beta) production and maturation of mRNA-transfected monocyte-derived dendritic cells.

BACKGROUND: The development of dendritic cell (DC)-based vaccines using antigen-encoding mRNA requires identification of the critical parameters for efficient ex vivo loading of DCs. Exogenously delivered mRNA can induce DC activation, but the molecular mechanisms involved are unknown. The aim of the present study was to identify the means by which mRNA-dependent activation of DCs occurs. METHODS: In vitro transcribed mRNA molecules were delivered into porcine monocyte-derived DCs (MoDCs) using different non-viral gene transfer procedures. Using the green fluorescent protein (GFP) as reporter gene, as well as rhodamine-labeled RNA, intracellular delivery and transfection efficiency were assessed by confocal microscopy and flow cytometry. DC activation was monitored in terms of MHC class II and CD80/86 upregulation, as well as the production of type I interferon (IFN-alpha/beta). RESULTS: mRNA-lipofected MoDCs produced type I IFN and upregulated MHC class II and CD80/86. Computational analysis of the mRNA molecules predicted highly ordered secondary structures forming double-stranded RNA (dsRNA). This dsRNA was also detectable by immunofluorescence in mRNA-lipofected cells, using antibody specific for dsRNA. Digestion of the mRNA prior to lipofection with a double-strand-specific RNase, but not a single-strand-specific RNase, abrogated DC activation. Impairment of protein kinase R (PKR) with 2-aminopurine also interfered with the activation. CONCLUSIONS: Double-stranded secondary structures on mRNA delivered by lipofection can activate MoDCs. This could have important implications for mRNA-based immunomodulation of DCs, DC-based immunotherapy, and formulation of RNA-based vaccines. In addition, this report describes the first in vitro steps towards development of a novel large animal model system to evaluate DC-based vaccines against infectious diseases.

Animals↗
PubMed1998

A urokinase receptor mRNA binding protein-mRNA interaction regulates receptor expression and function in human pleural mesothelioma cells.

Human pleural malignant mesothelioma (MS-1) or mesothelial (MeT5A) cells express the multifunctional urokinase receptor (uPAR) which influences neoplastic propagation via contributions to cellular proteolysis, migration, and mitogenesis. Recently, we reported that a 51-nucleotide fragment of the uPAR mRNA coding region contains regulatory information for uPAR message stability and that a cytoplasmic uPAR mRNA binding protein (uPAR mRNABp) specifically bound to this sequence in temporal association with uPAR mRNA destabilization in MS-1 cells. To determine if the uPAR mRNA-uPAR mRNABp interaction is a determinant of uPAR message stability as well as uPAR expression, we further characterized this cis-trans interaction and created stable transfected cell lines designed to exploit the interaction and to increase uPAR at the cell surface. The uPAR mRNABp was purified from MS-1 cells, has an apparent molecular mass of 50 kDa, selectively binds to the 51-nt fragment of the uPAR coding region, and does not degrade uPAR mRNA. To determine the role of the uPAR mRNABp on receptor expression, we overexpressed a chimeric beta-globin/uPAR/beta-globin mRNA containing the 51-nt binding fragment of uPAR mRNA in MS-1 cells and found that uPAR at the cell surface increased by twofold as measured by [125I]uPA binding or ligand blotting. Cellular proliferation of uPA-treated cells and invasiveness was similarly increased. The increase in cell surface uPAR was due to commensurately increased uPAR mRNA. The results suggest that competition between the overexpressed 51-nt fragment of the uPAR coding region and the wild-type uPAR mRNA transcript for uPAR mRNABp binding enables the cells to translate and express more uPAR at the cell surface. The interaction between the uPAR mRNABp and uPAR mRNA regulates message stability as well as uPAR expression by MS-1 cells.

Cytosol↗
PubMed1998

Cloning and characterization of three human cDNAs encoding mRNA (guanine-7-)-methyltransferase, an mRNA cap methylase.

The mRNA cap structure is synthesized by a series of reactions catalyzed by capping enzyme and mRNA (guanine-7-)-methyltransferase. mRNA (guanine-7-)-methyltransferase catalyzes the methylation of GpppN- at the guanine N7 position, which is an essential step for gene expression in eukaryotic cells. Here we isolated three human cDNAs encoding mRNA (guanine-7-)-methyltransferase termed hCMT1a, hCMT1b and hCMT1c. hCMT1a and hCMT1b encode 476 and 504 amino acids, respectively, and differ only at the region coding for the C-terminal portion of the enzyme after amino acid residue 465. The third cDNA hCMT1c seems to encode the same polypeptide as hCMT1a, however, the 3'-noncoding region of hCMT1c contains sequences corresponding to part of the C-terminal coding and noncoding regions of hCMT1b thus consisting of a mosaic of hCMT1a and hCMT1b. RT-PCR showed that all 3 types of mRNAs were expressed in every tissue examined. Comparison of the deduced amino acid sequences with those of other viral and cellular enzymes showed the regions which are highly conserved among mRNA (guanine-7-)-methyltransferases. The recombinant hCMT1a expressed in E. coli exhibited mRNA (guanine-7-)-methyltransferase activity. On the other hand, neither mRNA (guanine-7-)-methyltransferase nor mRNA (nucleoside-2'-O-)-methyltransferase activity was detected with the recombinant hCMT1b protein. Although the biological significance of the expression of these three mRNA (guanine-7-)-methyltransferase mRNA species remains unknown at present, the nucleotide sequences suggest that they are produced by alternative RNA splicing.

Amino Acid Sequence↗
PubMed2000

Glucose starvation reduces IGF-I mRNA in tumor cells: evidence for an effect on mRNA stability.

The purpose of this study was to characterize the mechanisms by which glucose regulates IGF-I gene expression in rat C6 glioma cells and in rat GH3 pituitary adenoma cells. Glucose starvation for periods of 12 to 48 h decreased IGF-I mRNA levels. In contrast, there was no stimulation of IGF-I mRNA by medium glucose between 1 and 25 mM over a 24-h period. Studies with hexoses and glycolytic metabolites suggested that glucose metabolism was required to maintain IGF-I mRNA. Glucose starvation lowered IGF-I mRNA half-life in both C6 and GH3 cells. Protein synthesis inhibition lowered IGF-I mRNA by about 20% in glucose-fed C6 and GH3 cells, while potently increasing IGF-I mRNA in glucose-starved C6 cells and not altering IGF-I mRNA in glucose-starved GH3 cells. Our results suggest that in these tumor cells, IGF-I mRNA stability is reduced by glucose starvation, secondary to a deficiency in intracellular glucose metabolism. Ongoing protein synthesis is not required for this mRNA de-stabilizing effect in GH3 cells. Rather, in glucose-starved C6 cells, decreased IGF-I mRNA stability may result from the action of a labile protein.

Animals↗
PubMed1995

Cell-cycle-dependent gene expression studied by two-colour fluorescent detection of a mRNA and histone mRNA.

We investigated whether a probe specific for histone H3 mRNA could be used as a marker to study cell-cycle dependency of gene expression by double-fluorescent RNA in situ hybridization (FISH). First, we showed that all S-phase cells in cell cultures having incorporated BrdU revealed histone H3 mRNA expression by RNA FISH, indicating that histone H3 expression is a reliable marker for S-phase cells. Second, we analysed whether the expression of human cytomegalovirus immediate early genes in rat 9G cells, which are known to be induced in an S-phase dependent way by cycloheximide, correlated with the expression of histone H3 mRNA. Double-hybridization experiments with a digoxigenin-labelled probe for IE mRNA and a fluoresceinated probe for histone H3 mRNA revealed that cells expressing IE mRNA also expressed histone H3 mRNA. Third, we examined the cell-cycle dependency of luciferase gene expression in X1 cells. Luciferase mRNA is heterogeneously expressed in X1 cell cultures, but cells expressing luciferase did not necessarily express histone H3 mRNA. This indicates that luciferase gene expression in X1 cells is not induced during S-phase. The results of our study show that histone H3 mRNA expression can be successfully used as a marker to establish cell-cycle dependency of gene expression by double-RNA FISH.

Animals↗