Search PubMed⌕ Search

Biomedical subjects

E Chu

Publications and source records attributed to E Chu.

At least 73 records · Page 4Linked to original sources
PubMed1993

Identification of an RNA binding site for human thymidylate synthase.

Previous studies from this laboratory have shown that human TS mRNA translation is regulated by its protein product in a negative autoregulatory manner. In this paper, we identify an RNA binding site for TS protein located within the first 188 nt of TS RNA. A 36-nt RNA sequence contained within this 188-nt fragment, corresponding to nt 75-110 and including the translational initiation site, binds TS protein with an affinity similar to that of both the full-length and the 188-nt TS RNA sequences. Variant RNAs with either a deletion or a mutation at the translational initiation region are unable to compete for TS protein binding. UV crosslinking studies reveal that an RNA fragment of approximately 36 nt is protected from RNase T1 digestion by TS protein binding. A second TS protein-binding site is localized within the protein-coding region corresponding to nt 434-634. These findings demonstrate a specific interaction between human TS protein and its TS RNA and identify an RNA binding site that includes the translational initiation site.

Base Sequence↗
PubMed1993

Suppression of the development of tumoricidal function in gamma interferon-treated human peripheral blood monocytes by lipopolysaccharide: the role of cyclooxygenase metabolites.

Bacterial lipopolysaccharide (LPS) is generally regarded as one of the most potent macrophage activators. Thus, LPS has been used as an obligatory second signal to stimulate macrophage cytotoxic function against a wide array of bacterial and neoplastic targets. In this study, however, we define conditions under which LPS can suppress the development of cytotoxic function in normal human peripheral blood monocytes. When monocytes were treated with a priming dose of gamma interferon (gamma-INF), followed 18-24 hr later by a triggering dose of LPS, significant cytotoxic function developed. However, when monocytes were treated with even minimal amounts of LPS during priming with interferon, the development of cytotoxic function following stimulation with a second, triggering dose of LPS was virtually abolished. This effect could be produced from 0 to 14 hr following the addition of gamma-INF. The inhibition of monocyte cytotoxicity which was produced by LPS treatment during priming was dose dependent and could not be overcome by modifying either the priming dose of gamma-IFN or the triggering dose of LPS. The suppression was largely overcome, however, by treatment with the cyclooxygenase inhibitor, indomethacin. The possibility that LPS-induced suppression of monocyte cytotoxicity was mediated by products of the cyclooxygenase pathway was supported further in this study by demonstrating that LPS stimulated the production of significant amounts of prostaglandin E2 (PGE2) from monocytes and that this was facilitated by gamma-IFN. In kinetics studies, it appeared that LPS suppression of monocyte activation was correlated temporally with a heightened sensitivity to suppression by exogenously added PGE2, a condition which was reduced greatly by the end of the priming phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Death↗
PubMed1993

Diagnostic considerations in patients with suspected coronary artery anomalies.

The diagnosis of coronary artery anomalies requires a high index of suspicion during the history and physical examination. Nonspecific presentations are common, although dramatic presentations such as myocardial infarction or aborted sudden death will occur. Typically the results of the physical examination and ECG and the chest x-ray film are normal except with ALCAPA or coronary-artery fistulas. Screening studies such as exercise stress testing have low sensitivity, and a negative evaluation does not rule out the presence of a significant anomaly. These important limitations must be considered. Echocardiography is a practical and frequently diagnostic test if specific attention is paid to the coronary arteries. This examination should be performed in most patients with suspect symptoms. Other noninvasive techniques such as nuclear and cine MRI cardiac imaging may become increasingly important, but their current use is anecdotal. Cardiac catheterization remains the gold standard; however, recognition of important clues and specific angiographic views are required to fully delineate many anomalies.

Adolescent↗
PubMed1993

Flavone acetic acid (FAA) with recombinant interleukin-2 (rIL-2) in advanced malignant melanoma. III: Cytokine studies.

Twelve patients undergoing IL-2 and flavone acetic acid (FAA) combination immunotherapy for advanced melanoma were studied throughout treatment for the induction of measurable levels of bioactive TNF, GM-CSF and IL-6 in their serum. This was to assess the extent of secondary cytokine induction in these patients and the possible role of such cytokines in both the toxic and therapeutic responses. The nature of the treatment schedule enabled these cytokines to be measured in response to FAA alone, FAA/IL-2 and FAA alone following IL-2/FAA activation of target cells. A small rise in the serum levels of these cytokines was seen on the initial course of FAA/IL-2 but this was minor compared to the marked elevation in levels 2-8 h following the initiation of the third course of FAA given with or without IL-2 and at a time point which coincided with maximum toxicity in those patients who experienced it. These results show that FAA alone can induce cytokine release from primed target cells. This may be associated with the therapeutic effect and/or toxicity of the agent.

Adult↗
PubMed1993

Curing reentrant atrial arrhythmias. Targeting protected zones of slow conduction by catheter ablation.

Radiofrequency catheter ablation has become the treatment of choice for paroxysmal supraventricular tachycardia involving dual atrioventricular nodes or an accessory pathway. For reentry confined to the atrium where the arrhythmia itself or the ventricular response cannot be controlled with drugs, catheter ablation of the His bundle is a treatment option, but requires implantation of a permanent pacemaker and does not restore normal rhythm. In the atria, important anatomic obstacles, such as the great veins and the ostium of the coronary sinus, interrupt the normal arrangement of myocardial fibers. Under certain circumstances these natural obstacles, or those created during atrial surgery for congenital heart disease, may help to facilitate conditions for reentrant excitation within the atrium. The purpose of this study was to evaluate the safety and efficacy of radiofrequency ablation directed at a protected isthmus of slow conduction in patients with reentrant atrial tachycardia or flutter. Eighteen patients with drug-refractory atrial arrhythmias underwent invasive electrophysiology testing, followed in the same session by ablation using radiofrequency energy delivered between the large distal electrode of a deflectable catheter and a skin patch. In eight patients, intracardiac echocardiographic imaging was performed to compliment fluoroscopy. These 18 patients had a total of 20 atrial tachyarrhythmias: atypical atrial flutter (1 patient), typical atrial flutter (13), intraatrial reentrant tachycardia (5), and sinus node reentry (1). There were 5 women and 13 men with an age range of 8 to 81 years. Structural heart disease was present in 10 of 14 patients with atrial flutter, and 4 patients with intraatrial reentrant tachycardias had surgery for congenital heart disease. Acute success was achieved in 12 of 13 cases (92%) of typical atrial flutter and in 6 of 6 cases of other atrial reentrant tachyarrhythmias, including sinus node reentry and five arrhythmias associated with congenital heart disease surgery. One patient developed a deep venous thrombosis. Radiofrequency catheter ablation, by severing narrow corridors of slow conduction, can safely abolish reentrant atrial arrhythmias in humans. Long-term follow-up evaluation will be required since these patients generally have atrial disease and recurrence of the ablated arrhythmia or the emergence of new arrhythmias is a possibility.

Adolescent↗
PubMed1993

Antimetabolites.

Explore the source record for details and available documents.

Antimetabolites, Antineoplastic↗
PubMed1993

Regulation of thymidylate synthase in human colon cancer cells treated with 5-fluorouracil and interferon-gamma.

The effects of fluorouracil (5-FU) and interferon-gamma (IFN-gamma) on the regulation of thymidylate synthase (TS) gene expression were investigated in the human colon cancer H630 cell line. By Western immunoblot analysis, TS protein levels in H630 cells were increased 3-, 5.5-, 5-, and 2.5-fold after 8-, 16-, 24-, and 36-hr exposure to 1 microM 5-FU, respectively. When H630 cells were exposed to varying concentrations of 5-FU (0.3-10 microM) for 24 hr, increases in TS protein up to 5.5-fold were observed. A 24-hr exposure to 1 microM 5-FU resulted in a 4.5-fold increase in the level of TS protein, whereas in 5-FU/IFN-gamma-treated cells TS protein was increased by only 1.8-fold, compared with control cells. IFN-gamma treatment alone did not affect TS protein levels, relative to control. Northern blot analysis revealed no changes in TS mRNA levels when H630 cells were exposed either to 1 microM 5-FU for 8-36 hr, to varying concentrations of 5-FU (0.3-10 microM) for 24 hr, or to the combination of 5-FU and IFN-gamma. Pulse-labeling studies with [35S]methionine demonstrated a 3.5-fold increase in net synthesis of TS in cells treated with 1 microM 5-FU, whereas the level of newly synthesized TS increased only 1.5-fold in cells treated with 5-FU/IFN-gamma, compared with control cells. Pulse-chase studies revealed that the half-lives of TS protein in control and 5-FU-treated cells were equivalent. These findings demonstrate that the increase in TS protein after 5-FU exposure and the subsequent inhibitory effect of IFN-gamma on TS protein expression are both regulated at the post-transcriptional level.

Colonic Neoplasms↗
PubMed1992

Enhancement of 5-fluoro-2'-deoxyuridine antineoplastic activity by 5-benzyloxybenzyloxybenzylacyclouridine in a human colon carcinoma cell line.

The pyrimidine acyclonucleoside benzyloxybenzyloxybenzylacyclouridine (BBBAU) showed growth inhibitory activity against the human colon cancer HCT-8 cell line with a 50% inhibitory concentration of 55 microM. Unlike its parent compounds, BBBAU was an extremely weak inhibitor of uridine phosphorylase. This acyclonucleoside analogue is an inhibitor of thymidylate synthase (TS) as determined by inhibition of [6-3H]-2'-deoxyuridine incorporation into DNA, inhibition of 3H release from [5-3H]-2'-deoxyuridine, and decrease in both the free and total TS 5'-fluoro-2'-deoxyuridine 5'-monophosphate binding sites. Kinetic analysis revealed that BBBAUMP, the monophosphate analogue of BBBAU, is a competitive inhibitor of purified human recombinant TS with a Ki of 8.0 microM. Nucleoside transport and uptake studies revealed that BBBAU (30 microM) inhibited the initial rate of transport and the total uptake of thymidine (25 microM). In contrast, while BBBAU (30 microM) inhibited the initial rate of transport of 5-fluoro-2'-deoxyuridine (FdUrd, 25 microM), its intracellular accumulation was increased. BBBAU (10 and 50 microM, respectively) potentiated FdUrd growth inhibition of HCT-8 cells and significantly enhanced the cytotoxic effects of FdUrd (0.3 and 1 microM, respectively) against HCT-8 cells using a clonogenic assay system. This combination resulted in additive inhibitory effects on TS activity resulting in greater depletion of dTTP pools. Moreover, the incorporation of radiolabeled FdUrd into the DNA fraction of HCT-8 cells was enhanced. The potential importance of this novel combination for human colon cancer chemotherapy is discussed.

Antimetabolites, Antineoplastic↗
PubMed1992

Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands.

Expression of the int-3 locus is activated in mouse mammary tumors as a consequence of insertional mutagenesis by the mouse mammary tumor virus (MMTV). Integration of the MMTV provirus into the int-3 locus promotes the transcription and translation of flanking cellular int-3 sequences sharing significant homology with the intracellular domain of the neurogenic Notch gene of Drosophila, and with the yeast cell cycle regulatory genes cdc10 and SWI6. To determine the in vivo consequences of activated int-3 expression, transgenic mice were generated harboring a genomic tumor DNA fragment consisting of the MMTV LTR and the flanking cellular int-3 sequences. All six int-3 founder transgenic mice and the progeny of one established line exhibited similar dramatic phenotypic abnormalities in tissues in which the transgene was expressed. Focal and often multiple poorly differentiated mammary and salivary adenocarcinomas appeared in the majority of transgenic mice between 2 and 7 months of age. Significantly, mammary glands were arrested in development and were lactation deficient in all female int-3 mice. The salivary glands, glands of the nasal mucosa and maxillary sinus, the extraorbital lacrimal glands, and the Harderian glands of juvenile and adult transgenic mice all contained proliferating immature ductule cells and were incompletely differentiated. In addition, all male int-3 transgenic mice were sterile, apparently the result of severe hyperplasia of the epididymis. These findings demonstrate in vivo that expression of the activated Notch-related int-3 gene causes deregulation of normal developmental controls and hyperproliferation of glandular epithelia.

Adenocarcinoma↗
PubMed1992

Antimetabolites.

Explore the source record for details and available documents.

Animals↗
PubMed1992

Biochemical modulation of fluorouracil with leucovorin and interferon: preclinical and clinical investigations.

Leucovorin and interferon are capable of modulating the cytotoxicity of fluorouracil (5-FU). Preclinical studies demonstrate that d,l-leucovorin is rapidly metabolized in human breast and colon cells into the various one-carbon substituted folate pools and to the polyglutamated state. While increases in intracellular folate pools are proportional to the exposure concentration of leucovorin, relatively large increases in leucovorin concentrations (50- to 100-fold) are required to produce small intracellular changes (twofold). Polyglutamation is favored by prolonged exposures to leucovorin. Polyglutamate forms have a prolonged intracellular retention and a higher affinity for the target enzyme, thymidylate synthase. Ratios of up to 20:1 inactive to active leucovorin stereo-isomers had essentially no effect on the intracellular metabolism of the active isomer. Interferon gamma interacts with 5-FU in H630 colon cancer cells at the level of thymidylate synthase and enhances cytotoxicity of 5-FU by eliminating the 5-FU-induced acute overexpression of the target enzyme. No alterations in the intracellular metabolism or nucleic acid incorporation of 5-FU could be demonstrated with the addition of interferon gamma. A clinical trial combining interferon-alfa-2a (IFN-alpha-2a) (subcutaneous days 1 to 7) with 5-FU and leucovorin (given IV days 2 to 6) demonstrated that these agents could be combined with acceptable toxicity. While the addition of interferon did not allow dose escalation of 5-FU, it resulted in a significant increase in drug exposure (1.5-fold) compared with matched cycles of 5-FU plus leucovorin without interferon. The overall response rate in this pilot study of 13 untreated patients with gastrointestinal adenocarcinoma was 46%, including two complete responses. There were no responses in eight patients who had previously failed therapy with 5-FU.

Colonic Neoplasms↗
PubMed1991

Autoregulation of human thymidylate synthase messenger RNA translation by thymidylate synthase.

Thymidylate synthase (TS; 5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45) is essential for the de novo synthesis of thymidylate, a precursor of DNA. Previous studies have shown that the cellular level of this protein is regulated at both the transcriptional and posttranscriptional levels. The regulation of human TS mRNA translation was studied in vitro with a rabbit reticulocyte lysate system. The addition of purified human recombinant TS protein to in vitro translation reactions inhibited translation of TS mRNA. This inhibition was specific in that recombinant TS protein had no effect on the in vitro translation of mRNA for human chromogranin A, human folate receptor, preplacental lactogen, or total yeast RNA. The inclusion of dUMP, 5-fluoro-dUMP, or 5,10-methylene-tetrahydrofolate in in vitro translation reactions completely relieved the inhibition of TS mRNA translation by TS protein. Gel retardation assays confirmed a specific interaction between TS protein and its corresponding mRNA but not with unrelated mRNAs, including human placenta, human beta-actin, and yeast tRNA. These studies suggest that translation of TS mRNA is controlled by its own protein end product, TS, in an autoregulatory manner.

Animals↗
PubMed1991

Induction of thymidylate synthase associated with multidrug resistance in human breast and colon cancer cell lines.

A series of Adriamycin-resistant human breast MCF-7 and human colon DLD-1 cancer cell lines were established by stepwise selection. The concentration of Adriamycin required to inhibit cell proliferation by 50% (IC50) in the parent breast line (MCF-7), Adriamycin-resistant lines (MCF-Ad5 and MCF-Ad10), and a 5-fluorouracil (5-FU)-revertant line (MCF-R) was 0.005, 3.3, 6, and 4.9 microM, respectively. The Adriamycin IC50 value for the resistant colon line (DLD-Ad) was 8.2 microM, 68-fold higher than that for its parent line (DLD-1) (IC50 = 0.12 microM). The MCF-Ad5 and MCF-Ad10 cells were cross-resistant to 5-FU, with respective 5-FU IC50 values of 11.7 and 22.5 microM, or 7.3- and 14-fold less sensitive than their parent MCF-7 (IC50 = 1.6 microM) line. The MCF-R line completely reverted in sensitivity to 5-FU, with an IC50 of 1.7 microM. The resistant DLD-Ad line was 3.5-fold more resistant to 5-FU than was the parent DLD-1 line. Using both the 5-fluoro-2'-deoxyuridine-5'-monophosphate binding and catalytic assays for measurement of thymidylate synthase (TS) activity, there was significantly increased TS activity in the resistant MCF-Ad5 (2.4- and 2.5-fold), MCF-Ad10 (11.5- and 6.8-fold), and DLD-Ad (4.8- and 10.7-fold) lines, for binding and catalytic assays, respectively, compared with their parent MCF-7 and DLD-1 lines. The level of TS in cytosolic extracts, as determined by Western immunoblot analysis, was markedly increased for the resistant MCF-Ad5 (31-fold), MCF-Ad10 (46-fold), and DLD-Ad (52-fold) cells. Measurement of TS mRNA levels by Northern analysis revealed elevation of TS mRNA in the resistant MCF-AD5 (16.7-fold), MCF-Ad10 (31-fold), and DLD-Ad (55-fold) cells. Southern analysis showed that this increase in TS mRNA was not accompanied by any major rearrangements or amplification of the TS gene. Incorporation of 5-FU into the RNA and DNA of the resistant MCF-Ad10 cells was not significantly different, compared with that for parent MCF-7 cells. These studies suggest that exposure of human breast and human colon cancer cells to Adriamycin leads to overexpression of TS, with concomitant development of resistance to 5-FU.

Blotting, Southern↗
PubMed1991

Antimetabolites.

Explore the source record for details and available documents.

Animals↗
PubMed1990

Interaction of gamma interferon and 5-fluorouracil in the H630 human colon carcinoma cell line.

The antiproliferative effects and pharmacological interactions of 5-fluorouracil (5-FU) in combination with gamma interferon (IFN-gamma) were determined against the human colon carcinoma H630 cell line in vitro. H630 was 9-fold more resistant to 5-FU, as compared to a relatively sensitive human colon line (C1). IFN-gamma showed modest antiproliferative activity against the H630 line, with a 50% inhibitory concentration of 440 units/ml. Simultaneous treatment of H630 with subinhibitory concentrations of IFN-gamma and 5-FU produced a significant enhancement of the 5-FU-associated growth inhibition. The growth-inhibitory activity of the combination against H630 was prevented by the addition of 20 microM thymidine. Thymidylate synthase (TS) activity was measured by both the 5-fluoro-2'-deoxyuridine-5'-monophosphate binding and catalytic assays, using cytosolic extracts. A 24-h exposure to 1 microM 5-FU in the H630 line resulted in a 3.1-fold increase in the total amount of TS, while in the 5-FU/IFN-gamma-treated cells TS remained unchanged from non-drug-treated control levels. Moreover, we found that free thymidylate synthase in the 5-FU/IFN-gamma-treated cells was significantly decreased, as compared to the cells treated with 5-FU alone. Incorporation of 5-FU into both the RNA and DNA fractions did not change with the addition of IFN-gamma. Accumulation of the fluoropyrimidine metabolites 5-fluoro-2'-deoxyuridine-5'-monophosphate and 5-fluorouridine-5'-triphosphate remained the same for 5-FU alone and the combination treatment. These findings suggest that acute TS induction by 5-FU may provide an important mechanism by which human colon carcinoma cells express decreased sensitivity to 5-FU and that IFN-gamma can reverse the development of resistance to 5-FU in the H630 line by inhibiting the overexpression of TS that results from 5-FU exposure. These studies contribute to a growing understanding of the complex interaction between 5-FU and IFN-gamma.

Adenocarcinoma↗
PubMed1990

Mechanism of thymidylate synthase inhibition by methotrexate in human neoplastic cell lines and normal human myeloid progenitor cells.

We have studied the roles of 5,10-methylenetetrahydrofolate (5,10-methylene-H4PteGlu) depletion and dihydrofolate (H2PteGlu) accumulation in the inhibition of de novo thymidylate synthesis by methotrexate (MTX) in human MCF-7 breast cancer cells. Using both a high pressure liquid chromatography system and a modification of the 5-fluoro-2'-deoxyuridine-5'-monophosphate radioenzymatic binding assay, we determined that the 5,10-methylene-H4PteGlu pool is 50-60% depleted in human MCF-7 breast cancer cells following exposure to 1 micron MTX for up to 21 h. Similar alterations in the 5,10-methylene-H4PteGlu pools were obtained when human promyelocytic HL-60 leukemia cells and normal human myeloid precursor cells were incubated with 1 micron MTX. The H2PteGlu pools within the MCF-7 cells increased significantly after 15 min of 1 micron MTX exposure, reaching maximal levels by 60 min. Thymidylate synthesis, as measured by labeled deoxyuridine incorporation into DNA, decreased to less than 20% of control activity within 30 min of 1 micron MTX exposure. The inhibition of thymidylate synthesis coincided temporally with the rapid intracellular accumulation of H2PteGlu, a known inhibitor of thymidylate synthase. Furthermore, inhibition of this pathway was associated in a log-linear fashion with the intracellular level of dihydrofolate. These studies provide further evidence that depletion of the thymidylate synthase substrate 5,10-methylene-H4PteGlu is inadequate to account completely for diminished thymidylate synthesis resulting from MTX treatment. Our findings suggest that acute inhibition of de novo thymidylate synthesis is a multifactorial process consisting of partial substrate depletion and direct enzymatic inhibition by H2PteGlu polyglutamates.

Breast Neoplasms↗
PubMed1990

Pulmonary hyperinfection syndrome with Strongyloides stercoralis.

A 65-year-old man with steroid-dependent chronic airflow obstruction presented with progressive dyspnea and weight loss. Travel history included a military tour in southeast Asia. A chest roentgenogram revealed hyperexpanded lung fields with diffusely increased interstitial markings. The Papanicolaou stain of expectorated sputum demonstrated the rhabditiform larvae of Strongyloides stercoralis. Endemic areas of infection include the southeastern United States, Puerto Rico, Central America, the Pacific basin, and central Africa. In recent immigrant groups and veterans of the Vietnam conflict, rates of infection are as high as 6 percent. The hyperinfection syndrome occurs in immunocompromised hosts and is associated with glucocorticoid steroid therapy. This allows massive proliferation of larval forms. Clinical clues include an appropriate travel history (even in the remote past), gastrointestinal symptoms, cutaneous symptoms, eosinophilia, or thrombocytosis. Our patient demonstrated a classic presentation of the hyperinfection syndrome, and the condition responded well to thiabendazole.

Aged↗