Biomedical subjects

G S Cox

Publications and source records attributed to G S Cox.

At least 19 recordsLinked to original sources

PubMed1994

Ultrasound-guided compression repair of postcatheterization pseudoaneurysms: results of treatment in one hundred cases.

PURPOSE: The purpose of this report is to describe the indications, technique, and results for ultrasound-guided compression repair (UGCR) of postcatheterization pseudoaneurysms at a large medical center in which catheter-based diagnostic and interventional procedures are frequently used. METHODS: We reviewed the initial series of 100 consecutive patients who underwent UGCR in our noninvasive vascular laboratory from May 1991 through August 1992. Nearly all (n = 95) of these pseudoaneurysms involved the common femoral artery or its major branches, and each was manually compressed with a 5 MHz linear transducer for 10-minute intervals until the pseudoaneurysm was completely occluded or the procedure was considered to be a failure. RESULTS: UCGR was immediately successful in 94 patients, including 30 (86%) of 35 patients who were receiving anticoagulants and 64 (98%) of the 65 who were not (p = 0.019). The average compression time to achieve occlusion was 33 minutes (range 10 to 120 minutes), but was nearly twice as long (51 minutes) for pseudoaneurysms that had been present for more than 14 days. There were no related complications, but recurrent pseudoaneurysms occurred in six (20%) of 30 patients who continued to receive formal anticoagulation, compared with only four (6%) of 64 who did not (p = 0.074). Eight of the 10 recurrences were discovered within 24 hours after primary UGCR, but two others presented at 16 and 35 days, respectively. Eight recurrent lesions were corrected by repeat UGCR, whereas the remaining two required surgical repair. CONCLUSIONS: UGCR provides a reliable alternative to surgical treatment for postcatheterization pseudoaneurysms. Adequate follow-up is important, however, especially in patients for whom continued anticoagulation is necessary.

Aged↗

PubMed1994

Permissive effect of cyclic AMP and cycloheximide for induction by sodium butyrate of the glycoprotein hormone alpha-subunit gene in choriocarcinoma cells.

Expression of the chorionic gonadotropin alpha-subunit (alpha CG) gene is regulated differently in human tumor cell lines derived from trophoblastic and nontrophoblastic tissues. This is based, at least in part, on the observations that production of alpha CG is increased by cAMP but not by sodium butyrate in choriocarcinoma cells (JEG-3), whereas production of alpha CG is increased by butyrate but not by cAMP in cervical carcinoma cells (HeLa). Data presented herein confirm that the steady-state levels of alpha CG mRNA are increased approximately 10-fold by cAMP in JEG-3 cells, but additionally demonstrate that these levels can be further increased (to 25-fold) by sodium butyrate in conjunction with the cyclic nucleotide. Similar effects were achieved with the phorbol ester 12-0-tetradecanoylphorbol-13-acetate, which also acted synergistically with cAMP to increase alpha CG mRNA levels (24-fold). Butyrate and 12-0-tetradecanoylphorbol-13-acetate had little or no effect when added alone (1.5-fold and 2.5-fold, respectively) or in combination with one another (3.8-fold). Induction of the alpha CG gene by cAMP was independent of protein synthesis, as mRNA levels were comparable when JEG-3 cells were treated with cAMP in the absence and presence of cycloheximide (CHX). Unexpectedly, alpha CG mRNA levels were also elevated 8- to 10-fold in response to a combination of butyrate and CHX, but CHX alone or in combination with 12-0-tetradecanoylphorbol-13-acetate had no effect. Gel mobility shift assays demonstrated changes in the pattern of proteins binding to a cAMP response element and to a trophoblast-specific element with nuclear extracts from cells treated with CHX but not with cAMP or sodium butyrate. Together, these results suggest that induction patterns of the alpha CG gene by butyrate and cAMP in trophoblast- and nontrophoblast-derived tumor cell lines are less distinct than suggested previously and indicate that CHX, which is permissive for induction by butyrate, causes a significant shift in electrophoretic mobility of trans-acting factors involved in alpha CG gene expression.

Base Sequence↗

PubMed1994

A new technique for left renal cryopreservation.

Our technique for left renal cryopreservation seems to have promise for preventing renal failure after thoracoabdominal aortic aneurysm repair. Based on the evidence we have obtained with this limited study, we plan to continue using this technique with selected, high-risk patients.

Aortic Aneurysm, Abdominal↗

PubMed1994

Reduction in the homologous blood requirement for abdominal aortic aneurysm repair by the use of preadmission autologous blood donation.

BACKGROUND: To evaluate the effectiveness of preadmission autologous blood donation (PABD) in reducing the homologous transfusion requirement of abdominal aortic aneurysm resection, the blood product requirements of 145 patients who underwent operation at Cleveland Clinic from September 1987 through May 1991 were reviewed. METHODS: A study group of 73 patients underwent aortic grafting for aneurysm after PABD. Intraoperative autotransfusion (IAT) was used routinely. Homologous blood requirements were compared to those of 72 patients at the same center who underwent similar operations using IAT alone. No significant differences were noted in age, gender, cardiovascular risk factors, operation complexity, intraoperative blood loss, or IAT volumes between the two groups. Mean aneurysm size of the study patients (5.4 cm) was slightly less than that of the comparison patients (6.0 cm) (p < or = 0.001). Patients in the study group received a mean of 1.9 units predeposited autologous blood. RESULTS: The mean discharge hematocrit (33.4%) and hemoglobin (11.0 gm/dl) levels of the study group were indistinguishable from those of the comparison group (33.3% and 11.1 gm/dl, respectively). The homologous blood requirement of the study group was significantly less (median, 0; mean, 1.3 units/patient) than that of the comparison group (median, 1.5; mean, 1.9 units/patient) (p = 0.001). Furthermore, 67% (49 of 73 patients) of the study group required no homologous blood although only 36% (26 of 72 patients) of the comparison patients avoided banked blood transfusions (p = 0.0004). No significant differences were found in platelet, fresh frozen plasma, or cryoprecipitate requirements between the study and comparison groups. CONCLUSIONS: PABD significantly reduces the homologous blood requirements for elective aortic aneurysm resection and, when used in combination with IAT, eliminates the need for homologous blood in at least two thirds of properly selected patients.

Aged↗

PubMed1993

Differential DNA methylation of the chorionic gonadotropin beta-subunit multigene family.

In addition to its synthesis in the developing placenta, human CG or the isolated alpha- and beta-subunits are synthesized by a wide variety of both trophoblastic and nontrophoblastic tumor cell lines. The beta-subunit confers unique biological activity on the hormone and is encoded by a family of six genes or pseudogenes linked physically with beta LH at one locus on chromosome 19. Previous work has demonstrated that members of the beta CG family are not expressed to the same extent in first-trimester placenta. The factors allowing differential expression of the six nearly identical genes are unknown. It is also undetermined which of the multiple beta CG genes are actively expressed in the tumor cell lines that produce beta-subunit ectopically. One potential mechanism for control of beta CG gene expression is DNA methylation. A unique method of two-dimensional DNA electrophoresis was used to analyze the methylation status of each of the individual beta CG genes in placenta and in tumor cell lines that either do or do not express the protein. The DNA from each source exhibited unique, reproducible methylation patterns over the six beta CG genes. Particularly interesting were the observations that: 1) despite their nearly identical sequences and close chromosomal proximity, the six beta CG genes are distinguished from one another within the same and different cell types by their extent and pattern of methylation; 2) the beta CG locus is significantly hypomethylated in placenta, a state maintained in choriocarcinoma cells (JAr); 3) the beta CG gene cluster is unmethylated at a limited set of sites in DNA from both 2RA (a transformed fibroblast cell line that does not produce beta-subunit) and CBT (a glioblastoma cell line that produces beta-subunit ectopically), suggesting that general demethylation of the domain does not accompany activation of the locus in CBT but rather that site-specific changes may be responsible, at least in part, for inappropriate beta CG expression; and 4) the ratio of beta CG gene 3 transcripts to beta CG gene 5 transcripts is at least 10-fold higher in CBT cells than in JAr cells, suggesting that the reduced methylation in CBT cells of gene 3 compared to gene 5 may be a factor contributing to the activity of these genes.

Base Sequence↗

PubMed1992

Thoracoabdominal aneurysm repair: a representative experience.

Between May 1966 and June 1991, 129 patients underwent surgical repair of thoracoabdominal aneurysms, with an overall 30-day mortality rate of 35%. In 75 operations (58%) performed electively, 11 deaths (15%) occurred, and in 54 cases (42%) of either symptomatic or ruptured aneurysms 34 deaths (63%; p less than 0.001) occurred. No one survived among six patients with preoperative hypotension (less than 90 mm Hg) or cardiac arrest. In 16 patients (12%) the etiology of aneurysms was a result of chronic aortic dissection, and the mortality rate in this subgroup was 44%. In the remaining 113 patients (88%) where the etiology was atherosclerosis, 38 deaths occurred (34%; p = 0.433). Spinal cord ischemia occurred in 25 cases (21%) among 116 patients who survived operation. Partial ischemia occurred in six cases (25%), and complete paraplegia occurred in the remainder. Complete and partial paraplegia occurred in 16 of 42 cases (38%) when all of the thoracic aorta was replaced (Crawford groups I, II) and in 9 of 74 cases (12%) when only the abdominal or lower thoracic aorta was replaced (Crawford groups III, IV; p = 0.016). Other complications included myocardial infarction (14 cases, 11%), respiratory failure (46 cases, 36%), and renal failure (33 cases, 27%). The major prospect for improved early survival of patients with thoracoabdominal aneurysms seems to be early detection and elective repair before the occurrence of symptoms.

Adult↗

PubMed1992

Deoxyribonuclease-hypersensitive sites in the glycoprotein hormone alpha-subunit gene from trophoblastic and nontrophoblastic human tumor cell lines: correlation with expression and effect of chemical inducers.

Human CG is composed of two subunits, alpha and beta. In addition to its eutopic synthesis in normal and malignant trophoblasts, the hormone is produced ectopically by a variety of tumor cell lines of nonplacental origin. Regulation of the alpha CG gene in trophoblasts appears to differ from that in nontrophoblasts. To determine whether these differences are reflected in the chromatin structure at the alpha CG locus, DNase I-hypersensitive sites within this domain were mapped in human tumor cell lines that differentially express the gene. Two hypersensitive sites were detected in DNA from cell lines that produce the alpha-subunit. The latter includes trophoblastic (JAr and JEG-3 choriocarcinoma) and nontrophoblastic (HeLa cervical carcinoma and ChaGo bronchogenic carcinoma) tumor cell lines. The most prominent site (HS 1) was located approximately 100 base pairs upstream from the transcription start site. In trophoblasts, accessibility of HS 1 increased substantially upon induction of the gene by cAMP, likely reflecting alterations in DNA-protein interactions at the cAMP response element and/or tissue-specific enhancer. In nontrophoblasts, where alpha-subunit synthesis is enhanced by sodium butyrate but not by cAMP, neither butyrate nor cAMP altered the accessibility of HS 1. The HS 2 is comprised of multiple sites with weak to moderate DNase sensitivity located downstream at +1600 to +4000 in cell lines that produce alpha-subunit. Cell lines that do not express the alpha CG gene possess a distinct hypersensitive site (HS 3) within the first intron at about +600; these include 3A-Sub-E (SV40 transformed placenta), CBT (glioblastoma multiforme), and CaSki (cervical carcinoma). Cleavage by DNase at HS 1 and HS 2 is not evident in nuclei from cell lines that do not produce alpha-subunit. These results suggest that HS 1 and HS 3 are characteristic of active and inactive states of the alpha CG gene, respectively, and that the accessibility of HS 1 generally correlates with the level of expression.

Cell Transformation, Neoplastic↗

PubMed1990

Effects of sodium butyrate and 5-azacytidine on DNA methylation in human tumor cell lines: variable response to drug treatment and withdrawal.

The effect of butyrate (Btr) and 5-azacytidine (azaC) on genomic DNA methylation was examined in a variety of human tumor cell lines. Butyrate treatment differentially affected 5-methylcytosine (m5C) content in a cell type-specific manner. For example, incubation of CBT cells (glioblastoma multiforme) for 48 h in medium containing 2 mM Btr resulted in the hypomethylation of their DNA; after removal of the drug, the m5C content was rapidly (24 h) restored to its original level. In contrast, when HeLa cells (cervical carcinoma) were cultured for 48 h in 3 mM Btr, their genomic DNA became more extensively methylated relative to the untreated control, and the hypermethylated state was maintained for long periods in culture. Treatment of HeLa cell lines with azaC resulted in extensive demethylation of their DNA. Following removal of the drug, the DNA was rapidly remethylated to levels equal to or greater than those of the untreated cells. The hypermethylated state has remained stable for over 180 generations. Together, the data suggest that cells respond rapidly to alter drug-induced reductions in the m5C content of their DNA, and this response can subsequently result in overmethylation. In contrast, when drug-induced hypermethylation of genomic DNA occurs, it may persist for many cell generations.

5-Methylcytosine↗

PubMed1990

Induction by cycloheximide of the glycoprotein hormone alpha-subunit gene in human tumor cell lines and identification of a possible negative regulatory factor.

The steady-state level of mRNA encoding the glycoprotein hormone alpha-subunit is increased about 4-fold in HeLa cells by cycloheximide (CHX) or puromycin at concentrations that inhibit protein synthesis. This effect is observed in a number of cell lines that ectopically produce alpha-subunit, including ChaGo (brochogenic carcinoma), FL (amnion), and HeLa (cervical carcinoma). No increase in alpha-subunit mRNA is evident in two choriocarcinoma cell lines (JAr, JEG-3) that produce alpha-subunit as an eutopic product. The half-life of alpha-subunit mRNA is unchanged in the presence of CHX, but nuclear run-on assays demonstrate a 2.6-fold greater loading of RNA polymerase on the alpha-subunit gene in nuclei from CHX-treated cells. These results suggest that inhibition of protein synthesis results in higher transcription rates and not in decreased mRNA turnover. A nuclear protein (Mr 50,000) that binds to a DNA fragment located 5' proximal to the alpha-subunit gene but not to more distal 5'-flanking sequence or to the alpha-subunit cDNA has been identified in HeLa but not in JEG-3 cell lines. The p50 DNA binding activity in HeLa cells decreases in the presence of CHX at a rate similar to that at which alpha-subunit mRNA increases. Moreover, in a series of HeLa cell clones, the levels of p50 are directly proportional to the magnitude of induction produced by CHX. These data are consistent with a model for alpha-subunit gene regulation involving a labile repressor and constitute yet another level of differential regulation of the alpha-subunit gene in cells that produce the hormone subunit in an ectopic versus eutopic manner.

Cell Line↗

PubMed1990

Molecular characterization of the Spirometra mansonoides genome: renaturation kinetics, methylation, and hybridization to human cDNA probes.

High molecular weight DNA from pleroceroid larvae of the tapeworm Spirometra mansonoides was purified from isolated nuclei by conventional techniques. The DNA so isolated has a melting temperature (Tm) of 87 degrees C and a guanine plus cytosine (G/C) content of 44%. 5-Methyl cytosine could not be detected in plerocercoid DNA by HPLC analysis of DNA hydrolysates, by radiolabeling 5'-termini of MspI digests with polynucleotide kinase, or by comparing restriction patterns generated by MspI and HpaII. Renaturation kinetics demonstrated that the genome of S. mansonoides contains repetitive as well as single copy sequences and has a genome size estimated at approx. 1.6 X 10(9) bp. Hybridization was carried out between plerocercoid DNA and cDNAs for human beta-actin, alpha-tubulin and growth hormone (hGH). Rationale for this analysis was based on known homologies among actin and tubulin genes in numerous species and on apparent similarities between hGH and a plerocercoid growth factor that may be reflected in similar DNA sequence. Scanning densitometry of dot blots demonstrated that the hGH probe annealed to the same extent at low stringency (1 M NaCl, 55 degrees C) to DNA from plerocercoids, rat liver and chicken erythrocytes; but this interaction was less than to DNA from human lymphocytes, calf thymus and mouse skin. Similar results were obtained when restriction endonuclease digests of these DNAs were analyzed by Southern transfer. Little or no hybridization of the growth hormone probe to plerocercoid DNA was evident at higher stringency (1 M NaCl, 65 degrees C). In contrast, human tubulin and actin probes showed extensive hybridization to pleroceroid restriction fragments under the high stringency conditions.

Actins↗

PubMed1990

Enhancement by theophylline of the butyrate-mediated induction of choriogonadotropin alpha-subunit in HeLa cells. I. Lack of correlation with cAMP.

The glycoprotein hormone common alpha-subunit can be induced in HeLa and other nontrophoblastic tumor cell lines by sodium butyrate. This report demonstrates that production of alpha-subunit can be further modulated by theophylline, especially in conjunction with butyrate. This synergism was not observed with other phosphodiesterase inhibitors such as xanthine, caffeine, theobromine, or methylisobutylxanthine. Induction by a combination of the short chain fatty acid plus the methylxanthine results from a decrease in the lag time after effector addition as well as a change in the rate of subunit accumulation. The increase in alpha-subunit is correlated with an increase in the levels of alpha-subunit mRNA, suggesting that induction is manifest at a pretranslational stage. The production of alpha-subunit was only marginally affected in cultures treated with 8-Br-cAMP or forskolin. Intracellular levels of cAMP were increased approximately threefold by methylisobutylxanthine, twofold by theophylline, fourfold by forskolin, and about 50% by butyrate, yet significant induction was achieved only by butyrate and theophylline. Taken together, these data suggest that the synergism between butyrate and theophylline is not mediated by cAMP.

Alkaline Phosphatase↗

PubMed1990

Enhancement by theophylline of the butyrate-mediated induction of choriogonadotropin alpha-subunit in HeLa cells. II. Effect of both agents on mRNA turnover.

In the accompanying paper it was demonstrated that among several methylxanthine phosphodiesterase inhibitors, only theophylline significantly increased production of the glycoprotein hormone alpha-subunit in HeLa cells, and that this action was synergistic with that of sodium butyrate. A correlation between alpha-subunit induction and cAMP concentrations was not evident. In this report we characterized the effect of these two drugs on the metabolism of alpha-subunit mRNA. Sodium butyrate decreased the apparent half-life of mRNAs encoding alpha-subunit, beta 2-microglobulin, and alpha-tubulin, as well as that of total poly(A)+ RNA and rRNA. Theophylline produced a two- to threefold increase in the apparent half-life of alpha-subunit mRNA but had no effect on the turnover of beta 2-microglobulin, alpha-tubulin, or total poly(A)+ mRNA. An inverse correlation was noted between the apparent half-life of the mRNA and the degree of destabilization elicited by butyrate. It is concluded that alpha-subunit induction by theophylline is in large part due to mRNA stabilization, and that the concerted effect of theophylline and butyrate results from inhibition by theophylline of the butyrate-mediated destabilization of alpha-subunit mRNA combined with the elevation in alpha-subunit gene transcription known to be produced by the fatty acid.

Blotting, Northern↗

PubMed1989

Chorionic gonadotropin synthesis by human tumor cell lines: examination of subunit accumulation, steady-state levels of mRNA, and gene structure.

A number of tumor cell lines have been examined that differentially produce human chorionic gonadotropin and the isolated alpha- or beta-subunits. It has been demonstrated that all of the cell lines studied to date contain genes for both alpha- and beta-subunits, indicating that differential and exclusive expression of one subunit is not the result of a particular cell line having lost the gene for the alternate subunit as a consequence of chromosome changes accompanying cell transformation. Because many of these established cell lines are aneuploid, it is also significant that no evidence was found for gene amplification in cell lines producing alpha-subunit at very high levels compared to those with very low level expression. Analysis of restriction endonuclease digests of tumor cell DNAs has demonstrated identical patterns for beta-subunit in KpnI digests and KpnI/HindIII double digests. Polymorphisms were observed for alpha-subunit in EcoRI and HindIII digests, but these did not correspond with expression of the alpha-subunit. Significant levels of either mRNA (as determined by dot blot and Northern transfer hybridization analysis) were accompanied by corresponding elevated levels of alpha- and beta-subunits (as determined by radioimmunoassay), suggesting that regulation of subunit production most likely occurs at a pretranslational stage. However, there were apparent differences in the relative ratio of alpha- and beta-subunits and their cognate mRNAs among the cell lines.

Chorionic Gonadotropin↗

PubMed1989

Modulation of glycoprotein hormone alpha-subunit levels, alkaline phosphatase activity, and DNA replication by antimetabolites in HeLa cultures.

Synthesis of the glycoprotein hormone common alpha-subunit and alkaline phosphatase (placental isozyme) has been examined in HeLa S3 cells. A variety of compounds that inhibit DNA synthesis lead to the increased production of both proteins. Experiments presented in this communication were undertaken to determine whether protein induction and DNA synthesis inhibition are coordinated. In general, nucleoside analogs and compounds that alter deoxynucleotide metabolism were good inducers of these ectopic products, whereas agents that altered DNA by intercalation, crosslinking, and covalent modification were poor inducers. The former class of effectors includes 5-bromo-2'-deoxyuridine, 5-fluoro-2'-deoxyuridine, 2'-deoxythymidine, 1-beta-D-arabinofuranosylcytosine, methotrexate, hydroxyurea, N-phosphonoacetyl-L-aspartic acid, and sodium butyrate; and the latter class of compounds includes ethidium bromide, acridine, bleomycin, mitomycin C, cesalin, macromomycin, and cis-diamminedichloroplatinum(II). A direct correlation between protein induction and DNA synthesis inhibition is unlikely based on the following observations: (i) for some effectors, the concentrations required to induce alpha-subunit and PAP were significantly different from those necessary to inhibit DNA synthesis; (ii) several agents inhibit DNA replication but do not enhance hormone or enzyme production; (iii) the kinetics of ectopic protein induction were similar for a number of inducers whereas the kinetics of DNA synthesis inhibition elicited by the same compounds were quite different. It is difficult from the data obtained, however, to rule out the possibility that inhibition of DNA synthesis may be required but is not sufficient for protein induction.

Alkaline Phosphatase↗

PubMed1988

Purification and characterization of the glycoprotein hormone alpha-subunit-like material secreted by HeLa cells.

The protein secreted by HeLa cells that cross-reacts with antiserum developed against the alpha-subunit of human chorionic gonadotropin (hCG) has been purified approximately 30,000-fold from concentrated culture medium by organic solvent fractionation followed by ion exchange, gel filtration, and lectin affinity chromatography. The final preparation had a specific activity (by RIA) of 6.8 x 10(5) ng of alpha/mg of protein and appeared homogeneous by electrophoresis on reducing/denaturing polyacrylamide gels (SDS-PAGE). Amino acid analysis indicated that HeLa-alpha had a composition very similar to that of the urinary hCG alpha-subunit. Peptide fingerprints of the HeLa protein and hCG-alpha revealed that several of the Tyr-, Met-, and Cys-containing tryptic peptides were held in common, thus identifying the tumor protein as a glycoprotein hormone alpha-subunit with a primary structure similar to that of hCG-alpha. However, comparison of hCG-alpha and HeLa-alpha demonstrated that the tumor-associated subunit was not identical with its normal counterpart. Only two of the three Tyr-containing tryptic peptides present in hCG-alpha could be detected in HeLa-alpha after iodination with 125I. HeLa-alpha eluted prior to hCG-alpha during Sephadex G-75 chromatography, but the subunits coeluted when the tumor protein was first subjected to mild acid hydrolysis. The purified tumor protein had an apparent molecular weight greater than that of the urinary alpha-subunit when analyzed by SDS-PAGE (Coomassie blue staining), and this difference was even greater when a partially purified preparation was examined by an immunoblot technique (Western). Isoelectric focusing of the HeLa and hCG subunits demonstrated that the tumor protein had a lower pI (4.7-5.5 compared to 6.5-7.8), and removal of sialic acid by mild acid hydrolysis did not entirely eliminate this difference. Immunoprecipitation and electrophoresis of alpha-subunit from HeLa cultures labeled with [3H]fucose indicated that the tumor subunit was fucosylated, whereas analysis of hCG-alpha hydrolysates by HPLC confirmed previous reports that the placental subunit does not contain fucose. HeLa alpha-subunit was unable to combine with hCG beta-subunit to form holo-hCG under conditions where the hCG alpha-subunit was able to do so. The results indicate that, regardless of whether or not a single alpha-subunit gene is being expressed in both normal and neoplastic tissues, posttranslational modifications lead to a highly altered subunit in the tumor. The differences observed may be useful in diagnosing neoplastic vs hyperplastic conditions and may lend insight into the mechanism of ectopic hormone production by tumors

Amino Acid Sequence↗

PubMed1988

Effect of pyrimidine deoxynucleosides and sodium butyrate on expression of the glycoprotein hormone alpha-subunit and placental alkaline phosphatase in HeLa cells.

Production of the glycoprotein hormone common alpha-subunit and placental alkaline phosphatase activity can be modulated in HeLa cells by a variety of deoxynucleosides. Dose response curves for thymidine (Thd), fluorodeoxyuridine (FdUrd), bromodeoxyuridine (BrdUrd) and iododeoxyuridine (IdUrd) demonstrate that, in general, alkaline phosphatase was increased by lower concentrations of inducer than was alpha-subunit. The deoxynucleosides were not as effective as sodium butyrate as inducers of either protein. Whereas Thd and the halogenated dUrd derivatives enhanced protein expression, deoxycytidine (dCyd) had negative effects. Induction by deoxynucleosides of both alkaline phosphatase and alpha-subunit was inhibited by dCyd, but induction of alkaline phosphatase by butyrate was more sensitive to dCyd inhibition than was the butyrate-mediated induction of alpha-subunit. These results suggest that the two proteins are not regulated in a coordinate manner. Reversal of alkaline phosphatase induction by dCyd was not observed in cells preincubated with sodium butyrate for 6-24 h before the addition of dCyd, indicating that the deoxynucleoside interferes with an early event in the butyrate-mediated response. Combinations of butyrate with Thd, BrdUrd or IdUrd were synergistic with respect to the induction of HeLa-alpha. It is concluded that incorporation of the deoxynucleosides into DNA may not be required for the synergistic response since 2',5'-dideoxythymidine was an effective as Thd. Cytoplasmic dot hybridizations demonstrate that a primary effect of the various effectors is to increase the steady-state levels of alpha-subunit mRNA. There was a good correlation between alpha-subunit accumulation and corresponding levels of alpha-mRNA, suggesting that regulation occurs at a pretranslational site. Although the mechanism(s) is not understood, these data provide evidence that nucleosides or their derivatives can significantly affect gene expression.

Alkaline Phosphatase↗

PubMed1987

Dual effects of 2-deoxyglucose on synthesis of the glycoprotein hormone common alpha-subunit in butyrate-treated HeLa cells.

Sodium butyrate (Btr) (3 mM) causes a 10-fold increase in production of the glycoprotein hormone alpha-subunit in HeLa cells. The following report demonstrates that this response could be inhibited about 95% by 5 mM 2-deoxy-D-glucose (dGlc), whereas alpha-subunit production in uninduced cells was affected little or not at all. Addition of D-mannose restored the Btr induction of Hela-alpha in cultures that had been treated with dGlc. When the alpha-subunits secreted by cells cultured in Btr plus dGlc or in Btr alone were compared by gel filtration (Sephadex G-75) and lectin affinity (concanavalin A and ricin) chromatography, differences were noted that probably reflect changes in their carbohydrate moieties. Immunoprecipitation of [35S]methionine-labeled HeLa-alpha and incubation with endoglycosidase H indicated that the subunit secreted from cells in the presence of dGlc contained oligosaccharide side chains that were not processed to the complex type. Cells that were simultaneously treated with Btr plus dGlc showed no increase in alpha-subunit production over cells receiving Btr only; in contrast, cells that were preincubated with Btr for either 16 or 36 h before dGlc was added exhibited high levels of subunit synthesis. Measurement of alpha-mRNA levels at various times after Btr and dGlc were added to cultures indicated that Btr brought about a dramatic increase in alpha-specific mRNA about 24 h after being added to cultures. This increase could be prevented by dGlc when added simultaneously with Btr but not when added after a 24-h preincubation. Although dGlc prevented the induction of alpha-subunit and alpha-mRNA in response to Btr, it had no effect on histone hyperacetylation, suggesting that if this chromatin modification is necessary for the induction process, it is not in itself sufficient. Together, the data demonstrate that dGlc inhibits the accumulation of alpha-subunit mRNA normally produced in response to Btr and that the subunit produced contains altered oligosaccharide constituents.

Butyrates↗

PubMed1986

Phosphorylation of the glycoprotein hormone alpha-subunit secreted by human tumor cell lines.

The synthesis of ectopic proteins by tumors is thought to result from derepression of normally silent genes. One approach to a better understanding of this phenomenon is to characterize the physicochemical properties of the ectopic products, comparing them to their normal counterparts. In the following communication, evidence will be presented to indicate that the glycoprotein hormone alpha-subunits secreted by a number of human tumor cell lines are phosphorylated. This novel covalent modification occurs in cell lines derived from both trophoblastic (JAR, JEG) and nontrophoblastic (HeLa, ChaGo) tumors. A choriocarcinoma cell line (JAR), which secretes both hCG-alpha and hCG-beta, phosphorylates only the alpha-subunit.

Autoradiography↗