Identification of Alu transposition in human lung carcinoma cells: a correction.
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Carcinoma↗
Biomedical subjects
D Samols
Publications and source records attributed to D Samols.
Control of the expression of c-sis mRNA in human glioblastoma cells by phorbol ester and transforming growth factor beta 1.
The regulation of c-sis oncogene expression in human glioblastoma cell line A172 has been investigated using a sensitive RNA-RNA solution hybridization method. Enhanced expression of c-sis mRNA was induced by phorbol ester (PMA) and diacylglycerol, each of which activates protein kinase C. c-sis mRNA was also induced by transforming growth factor beta (TGF-beta). The response to PMA and TGF-beta was transient, and in each case the decrease in c-sis mRNA level following maximum stimulation occurred with a half-life similar to the mRNA half-life previously determined. Cycloheximide had no significant effect on the induction of c-sis mRNA by either PMA or TGF-beta. The increases in c-sis mRNA following addition of either PMA or TGF-beta correlated well with increases in c-sis transcription as observed by the nuclear run-on technique. In cells in which protein kinase C had been down-regulated, there was no inhibition of the c-sis mRNA response to TGF-beta. Furthermore in cells pretreated with TGF-beta, induction by PMA was unaffected. Thus the TGF-beta signal pathway does not involve activation of protein kinase C, and at least two initially distinct intracellular signaling routes lead to activation of c-sis gene expression in this glioblastoma cell line. The protein kinase inhibitor H7 abolished the ability of not only PMA but also of TGF-beta to induce c-sis mRNA. The ability of H7 to inhibit the TGF-beta stimulation suggests that a protein kinase other than protein kinase C is involved in the signal transduction by TGF-beta.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Major structural alterations of the c-sis gene are not observed in a series of tumors of the human central nervous system.
Expression of the c-sis oncogene, the gene encoding the B chain of platelet-derived growth factor (PDGF), may be related to initiation and/or progression of glial cell tumorigenesis by PDGF-mediated autocrine growth stimulation. As the mechanism for activation of expression of the c-sis gene in gliomas is not known, we searched for possible structural alterations of c-sis DNA in these tumors. Genomic Southern blots of DNA from 7 different cultured human glioblastoma cell lines and 15 different solid human brain tumors revealed no significant change in either the gross structure or the copy number of the c-sis gene in tumor cells vs. control cells. Activation of glioma c-sis gene expression is therefore not the result of a gross rearrangement or amplification of the c-sis gene. Expression of c-sis mRNA was detected in all of 12 different solid human brain tumors, 11 of which were of glial cell origin. However, in tissue adjacent to 5 different tumors, approximately the same level of c-sis mRNA was seen.
Brain Neoplasms↗
A rapid procedure to identify newborn transgenic mice.
We have developed a rapid procedure to identify newborn transgenic mice containing foreign genetic material in their genome. The protocol involves collagenase digestion of a small amount of tail tissue which can be taken very early after birth, phenol and chloroform extraction, polymerase chain reaction, and polyacrylamide gel electrophoresis. The entire procedure, from tissue biopsy to final results, can be completed in 1 day.
Animals↗
Regulation of rabbit acute phase protein biosynthesis by monokines.
We defined the acute phase behaviour of a number of rabbit plasma proteins in studies (in vivo) and studied the effects of monokine preparations on their synthesis by rabbit primary hepatocyte cultures. Following turpentine injection, increased serum levels of C-reactive protein, serum amyloid A protein, haptoglobin, ceruloplasmin, and decreased concentrations of albumin were observed. In contrast to what is observed in man, concentrations of alpha 2-macroglobulin and transferrin were increased. Co-culture of primary hepatocyte cultures with lipopolysaccharide-activated human peripheral blood monocytes or incubation with conditioned medium prepared from lipopolysaccharide-activated human or rabbit monocytes resulted in dose-dependent induction of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and depression of albumin synthesis, while C-reactive protein synthesis and mRNA levels remained unchanged. A variety of interleukin-1 preparations induced dose-dependent increases in the synthesis and secretion of serum amyloid A, haptoglobin, ceruloplasmin and transferrin and decreased albumin synthesis. Human recombinant tumour necrosis factor (cachectin) induced a dose-dependent increase in synthesis of haptoglobin and ceruloplasmin. In general, human interleukin-1 was more potent than mouse interleukin-1 and tumour necrosis factor. None of the monokines we studied had an effect on C-reactive protein synthesis or mRNA levels. These data confirm that C-reactive protein, serum amyloid A, haptoglobin and ceruloplasmin display acute phase behaviour in the rabbit, and demonstrate that, in contrast to their behaviour in man, alpha 2M and transferrin are positive acute phase proteins in this species. While both interleukin-1 and tumour necrosis factor regulate biosynthesis of a number of these acute phase proteins in rabbit primary hepatocyte cultures, neither of these monokines induced C-reactive protein synthesis. Comparison of these findings with those in human hepatoma cell lines, in which interleukin-1 does not induce serum amyloid A synthesis, suggests that the effect of interleukin-1 on serum amyloid A synthesis may be indirect.
Acute-Phase Proteins↗
Expression and stability of c-sis mRNA in human glioblastoma cells.
The production of platelet-derived growth factor like (PDGF-like) material by glioblastomas may be involved in the conversion of normal cells to tumor cells. In an investigation of this problem, we have examined some of the properties of the platelet-derived growth factor B-chain mRNA (c-sis mRNA) by a sensitive and quantitative RNA-RNA solution hybridization method. In 5 out of 8 human glioblastoma cell lines, c-sis mRNA was present, and in the line with the highest level, there were approximately 4-10 molecules per cell. The half-lives of the c-sis mRNA in two glioblastoma cell lines were 2.6 and 3.4 h, while in human umbilical vein endothelial (HUVE) and bladder carcinoma (T24) cells they were 1.6 and 2.5 h, respectively. Inhibiting protein synthesis produced no significant alteration of the c-sis mRNA half-lives in the glioblastoma or HUVE cells. The A-U-rich sequence at the 3' end of the c-sis mRNA therefore does not appear to affect the mRNA stability in the presence of cycloheximide as it does in other transcripts. The similarity of the c-sis mRNA half-lives in normal and tumor cells suggests that regulation of stability of c-sis mRNA is not a major factor in tumorigenesis in the glioblastoma cell lines examined.
Cycloheximide↗
Identification of Alu transposition in human lung carcinoma cells.
We have demonstrated genetic transposition in human cells. An experimental system was established in which the Ecogpt (gpt) gene was employed as a target for inactivation. The human lung carcinoma cell line A549 containing this target was fused to UV-irradiated A549 cells that did not contain the target. From the fusion products, sublines carrying an inactivated gpt gene were analyzed. UV irradiation increased the frequency of inactivated gpt genes in the fusion cells by 100-fold. One subline was found to contain a complete Alu sequence in the coding region of the gpt gene. The inserted element differed from the Blur8 sequence by only 7 out of the 270 nucleotides. The insertion of this Alu element created a 5 bp insertion site duplication.
Autoradiography↗
Heterogeneous nature of the acute phase response. Differential regulation of human serum amyloid A, C-reactive protein, and other acute phase proteins by cytokines in Hep 3B cells.
Because a number of different cytokines have been reported to regulate the synthesis of human, murine, and rat acute phase proteins (APP), we studied the effect of cytokines on production of several major human APP in a single system, the human hepatoma cell line Hep 3B. Conditioned medium (CM) prepared from human blood monocytes activated with LPS in the presence of dexamethasone led to substantial induction of serum amyloid A (SAA) and C-reactive protein (CRP) synthesis whereas the defined cytokines IL-1 beta, TNF alpha, and medium from a human keratinocyte cell line (COLO-16), containing hepatocyte-stimulating factor activity, failed to induce these two major APP. Induction of SAA and CRP was accompanied by an increase in concentration of their specific mRNA. Size fractionation of CM from activated monocytes by fast protein liquid chromatography indicated that SAA- and CRP-inducing activity eluted as a single peak with a Mr of approximately 18 kDa. alpha 1-Antitrypsin, which also failed to respond to IL-1 beta or TNF alpha, was induced by both CM and medium from COLO-16 cells. The induction of AT by CM was accompanied by an increase in specific mRNA. Induction of ceruloplasmin and alpha 1-antichymotrypsin and decrease in the synthesis of albumin was achieved by both CM and IL-1 beta. Ceruloplasmin and albumin responded in a comparable fashion to both TNF alpha and medium from COLO-16 cells; the response of ACT to these cytokines was not evaluated. These results indicate that human SAA and CRP are induced in Hep 3B cells by products of activated monocytes but not by IL-1 beta, TNF-alpha, or some hepatocyte-stimulating factor preparations and that a group of heterogeneous mechanisms are involved in the induction of the various human APP.
Acute-Phase Proteins↗
Evolutionary conservation among biotin enzymes.
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Acetyl-CoA Carboxylase↗
Secretion of rabbit C-reactive protein by transfected human cell lines is more rapid than by cultured rabbit hepatocytes.
C-reactive protein (CRP) is a major acute phase protein in humans and rabbits. Its synthesis by the liver varies over a 1000-fold range depending on the presence and severity of inflammatory stimuli. In previous studies of synthesis and secretion of rabbit CRP, we showed that secretion becomes more efficient over the course of the acute phase response as CRP synthesis rates increase (Macintyre, S.S., Kushner, I., and Samols, D. (1985) J. Biol. Chem. 260, 4169-4173). The current studies were undertaken to help distinguish between two alternative explanations for this finding: 1) that secretion efficiency may simply be a property of the rate of synthesis and intracellular concentration of CRP or 2) that secretion may be regulated by separate intracellular mechanisms. A fusion gene containing the mouse metallothionein I promoter linked to the protein coding region of the rabbit CRP gene was introduced into the human hepatoma cell line, NPLC, and the nonliver cell line, HeLa. In this system a graded response of the mouse metallothionein I promoter following exposure to increasing zinc concentrations results in increasing CRP synthesis. Unlike hepatocytes from rabbits undergoing the acute phase response, we found that rabbit CRP was secreted by these transfected cell lines with a very high degree of efficiency which was independent of the rate of CRP synthesis. This finding implies that normal rabbit hepatocytes retard the secretion of CRP and that this inhibition is diminished as the acute phase response progresses. It further indicates that the relationship between changes in synthetic rate and efficiency of secretion of rabbit CRP is not a causal one and that synthesis and secretion of CRP by rabbit hepatocytes are regulated by independent intracellular mechanisms during the acute phase response.
Animals↗
Cloning and cDNA sequence of the dihydrolipoamide dehydrogenase component human alpha-ketoacid dehydrogenase complexes.
cDNA clones comprising the entire coding region for human dihydrolipoamide dehydrogenase (dihydrolipoamide:NAD+ oxidoreductase, EC 1.8.1.4) have been isolated from a human liver cDNA library. The cDNA sequence of the largest clone consisted of 2082 base pairs and contained a 1527-base open reading frame that encodes a precursor dihydrolipoamide dehydrogenase of 509 amino acid residues. The first 35-amino acid residues of the open reading frame probably correspond to a typical mitochondrial import leader sequence. The predicted amino acid sequence of the mature protein, starting at the residue number 36 of the open reading frame, is almost identical (greater than 98% homology) with the known partial amino acid sequence of the pig heart dihydrolipoamide dehydrogenase. The cDNA clone also contains a 3' untranslated region of 505 bases with an unusual polyadenylylation signal (TATAAA) and a short poly(A) track. By blot-hybridization analysis with the cDNA as probe, two mRNAs, 2.2 and 2.4 kilobases in size, have been detected in human tissues and fibroblasts, whereas only one mRNA (2.4 kilobases) was detected in rat tissues.
Amino Acid Sequence↗
Effect of mutations at Met-88 and Met-90 on the biotination of Lys-89 of the apo 1.3S subunit of transcarboxylase.
The apo 1.3S subunit of transcarboxylase contains the sequence Ala-87-Met-88-Lys-89-Met-90, and it is Lys-89 that is biotinated. This sequence is highly conserved in all the biotin enzymes that have been sequenced (with the exception of acetyl-CoA carboxylase from chicken liver, which has Val in place of Ala). The role of Met-88 and Met-90 in specifying Lys-89 for biotination by synthetase was examined by site-directed mutagenesis. Genes of the 1.3S subunit coding for Thr-88, Leu-88, or Leu-90 were generated by oligonucleotide-directed in vitro mutagenesis and expressed in Escherichia coli. The mutated apo 1.3S subunits were isolated and the biotination by homogeneous synthetase from Propionibacterium shermanii was compared with that of the apo wild-type subunit. The Vmax for the apo mutants was the same as that for the apo wild type, but when Leu was substituted for Met-88 or Met-90, the Km for the mutant was lower than that of the wild-type or mutant Thr-88. The activity of the synthetase of E. coli was determined by an in vivo assay. During the early log phase of growth, a smaller portion of mutants Thr-88 and Leu-90 was biotinated than with the wild-type or mutant Leu-88. When the cultures progressed to stationary phase, mutants and the wild type were biotinated to the same extent. The overall results show that Met-88 and Met-90 are not required for biotination of the apo 1.3S subunit by the synthetases.
Amino Acid Sequence↗
Cloning of rat brain succinyl-CoA:3-oxoacid CoA-transferase cDNA. Regulation of the mRNA in different rat tissues and during brain development.
3-Oxoacid CoA-transferase, which catalyses the first committed step in the oxidation of ketone bodies, is uniquely regulated in developing rat brain. Changes in 3-oxoacid CoA-transferase activity in rat brain during the postnatal period are due to changes in the relative rate of synthesis of the enzyme. To study the regulation of this enzyme, we identified, with a specific polyclonal rabbit anti-(rat 3-oxoacid CoA-transferase), two positive cDNA clones (approx. 800 bp) in a lambda gtll expression library, constructed from poly(A)+ RNA from brains of 12-day-old rats. One of these clones (lambda CoA3) was subcloned into M13mp18 and subjected to further characterization. Labelled single-stranded probes prepared by primer extension of the M13mp18 recombinant hybridized to a 3.6 kb mRNA. Rat brain mRNA enriched by polysome immunoadsorption for a single protein of size 60 kDa which corresponds to the precursor form of 3-oxoacid CoA-transferase was also found to be similarly enriched for the hybridizable 3.6 kb mRNA complementary to lambda CoA3. Affinity-selected antibody to the lambda CoA3 fusion protein inhibited 3-oxoacid CoA-transferase activity present in rat brain mitochondrial extracts. The 3.6 kb mRNA for 3-oxoacid CoA-transferase was present in relative abundance in rat kidney and heart, to a lesser extent in suckling brain and mammary gland and negligible in the liver. The specific mRNA was also found to be 3-fold more abundant in the brain from 12-day-old rats as compared with 18-day-old foetuses and adult rats, corresponding to the enzyme activity and relative rate of synthesis profile during development. These data suggest that 3-oxoacid CoA-transferase enzyme activity is regulated at a pretranslational level.
Animals↗
Mutagenesis affecting the carboxyl terminus of the biotinyl subunit of transcarboxylase. Effects on biotination.
Biotin is added to biotin-containing enzymes as a post-translational modification catalyzed by holoenzyme synthetase. This reaction is fairly general in that synthetase from one organism will modify enzymes from heterologous sources. This suggests that the polypeptides share some structural characteristic(s) that define(s) them as biotin enzymes. We have reported previously that when the gene coding for the 1.3 S biotinyl subunit of transcarboxylase is expressed in Escherichia coli, the polypeptide produced is biotinated by the cellular synthetase. Using in vitro mutagenesis of this gene, we have begun to define the primary structure involved in the enzymatic addition of biotin to a lysine residue. We show here that the carboxyl terminus of the 1.3 S subunit is critical in biotination. Mutations affecting the COOH-terminal residue do not influence the modification, but elimination of the hydrophobic side chain of the penultimate residue abolishes biotin addition.
Amino Acid Sequence↗
Cloning and characterization of the gene for rabbit C-reactive protein.
C-reactive protein (CRP), an acute-phase plasma protein of hepatic origin in man and rabbit, is a cyclic pentamer composed of five identical nonglycosylated Mr 22 500 subunits. We have isolated both cDNA and genomic clones for rabbit CRP. These clones were used as probes to demonstrate that when CRP synthesis is increased following an acute inflammatory stimulus, there is a corresponding increase in the level of accumulated CRP mRNA. The rabbit CRP gene is 2.6 kilobases in length containing a single intron of 252 base pairs (bp) which interrupts the codon for amino acid 2 in the protein. The mRNA for CRP contains a 5'-nontranslated region of 113 bp and a 3'-nontranslated region of 1550 bp. Sequencing of the protein-coding region of the gene indicates that the primary translation product contains a 20 amino acid N-terminal signal peptide. The deduced amino acid sequence is in general agreement with the published sequence [Wang, C. M., Nguyen, N. Y., Yonaha, K., Robey, F., & Liu, T.-Y. (1982) J. Biol. Chem. 257, 13610-13615] except in the region between amino acids 63 and 73. In this region, the sequence of both cDNA and genomic clones indicates the presence of 28 amino acids not previously reported. This alteration may be the result of genetic heterogeneity or an error in the reported protein sequence.
Amino Acid Sequence↗
Studies of translatable mRNA for rabbit C-reactive protein.
C-reactive protein (CRP) mRNA was assayed by cell-free translation of poly(A)-containing liver RNA isolated both from rabbits stimulated to undergo the acute-phase response and from unstimulated control rabbits. No CRP-related translation products were identified until the denaturant methylmercury hydroxide (CH3HgOH) was added to the RNA before cell-free translation. In the presence of the denaturant, a 24000-Da translation product was synthesized which was immunochemically identifiable as the CRP primary translation product. It is likely that rabbit CRP mRNA can form a stable intramolecular duplex which interferes with its translatability in vitro. The 24000-Da CH3HgOH-facilitated cell-free translation product was not detected in poly(A)-containing liver RNA from unstimulated animals, indicating that the concentration of translatable CRP mRNA was dramatically induced during the acute-phase response. On the basis of absorption experiments, the 24000-Da CRP primary translation product was immunochemically more closely related to denatured CRP than to native CRP.
Animals↗
Secretion of C-reactive protein becomes more efficient during the course of the acute phase response.
We studied the kinetics of synthesis and secretion of the acute phase plasma protein, C-reactive protein, in primary hepatocyte cultures prepared from rabbits manifesting differing degrees of the acute phase response to inflammatory stimulus. In cultures prepared from progressively more responsive animals, rate of C-reactive protein secretion increased to a much greater degree than did intracellular C-reactive protein content, resulting in a progressive decrease in the ratio of intracellular content to rate of secretion. This ratio, which represents the time required to secrete the amount of C-reactive protein contained within the intracellular pool, decreased from 18 h in cultures from unstimulated rabbits to 2.5 h in cells from highly responsive animals. In contrast, these ratios for albumin were short and fell within a narrow range (0.8-2.1 h). In pulse-chase labeling experiments, the time required for secretion of 50% of pulse-labeled C-reactive protein varied markedly, ranging from well over 6 h in cells from a minimally responsive animal to about 75 min in cells from a highly responsive rabbit. In contrast, the half-time for secretion of albumin was consistently about 45 min in the same cultures. Taken together, these findings indicate that the process by which C-reactive protein is secreted becomes more efficient during the course of the acute phase response. Recent studies have indicated that secretory proteins pass from the rough endoplasmic reticulum to Golgi at different and characteristic rates, possibly by a receptor-mediated process in which rate of transfer is determined by receptor affinity. We postulate that C-reactive protein secretion is regulated, during the course of the acute phase response, either by alterations in availability of specific receptors or by competition between different secretory proteins for a common receptor.
Animals↗
Cloning and expression of the 1.3S biotin-containing subunit of transcarboxylase.
We have cloned the gene coding for the 1.3S biotin-containing subunit of transcarboxylase (EC 2.1.3.1) from Propionibacterium shermanii. Transcarboxylase is a well-characterized enzyme composed of 30 polypeptides of three different types: twelve 1.3S biotinyl subunits, six 5S dimeric outer subunits, and one 12S hexameric central subunit. In propionic acid fermentation, the enzyme catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate in two partial reactions. The 1.3S subunit binds the outer and central subunits of the enzyme together, and its biotin serves as carboxyl carrier between subsites on the central and outer subunits where each partial reaction occurs. The cloned gene has been expressed in Escherichia coli, and the 1.3S subunit accumulates to 7% of total cellular protein. The foreign protein is recognized and biotinated by biotin holoenzyme synthetase of E. coli. The identifications of the gene and its product were confirmed by four independent approaches: DNA sequence analysis, immunoprecipitation, incorporation of labeled biotin, and measurement of enzymatic activity in the first partial reaction.
Amino Acid Sequence↗