Mechanisms of clinical resistance to 5-fluorouracil chemotherapy.
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Animals↗
Biomedical subjects
E Chu
Publications and source records attributed to E Chu.
The role of thymidylate synthase in cellular regulation.
Thymidylate synthase plays a central role in the biosynthesis of thymidylate, an essential precursor for DNA biosynthesis. In addition to its role in catalysis and cellular metabolism, studies from our laboratory have shown that thymidylate synthase functions as an RNA binding protein. Specifically, thymidylate synthase binds with high affinity to its own mRNA resulting in translational repression. An extensive series of experiments have now been performed to elucidate the molecular elements underlying the interaction between thymidylate synthase and its own mRNA. These studies have shed new light into the critical nucleotide sequences and/or secondary structure that are important for protein recognition. As well, studies to define the domains on the protein essential for RNA binding are currently underway. In addition to the characterization of the cis- and trans-acting elements underlying the interaction between thymidylate synthase and its own mRNA, we have recently shown that thymidylate synthase has the capacity to specifically bind in vitro and in vivo to other cellular RNA species. In this regard, thymidylate synthase interacts with the mRNAs of the c-myc onocogene and the p53 tumor suppressor gene. These two genes have been shown to play critical roles in cell cycle control, DNA biosynthesis, and apoptosis. In vitro studies reveal that the interaction of TS with these cell-cycle related mRNAs results in their translational repression. While the biological significance of these cellular RNA/TS protein interactions remains to be defined, these studies suggest a potential role for TS as a mediator in the coordinate regulation of several critical aspects of cellular metabolism.
Antineoplastic Agents↗
New Concepts for the Development and Use of Antifolates.
Approximately one-third of all cases of colorectal carcinoma present in an advanced and, therefore, incurable stage. For these patients, the development of new chemotherapeutic strategies is of central importance. Biochemical modulation of 5-fluorouracil (5-FU) has resulted in approximately a twofold increase in activity of 5-FU. Recent preclinical investigations suggest that interferon can also modulate the activity of 5-FU and may result in enhanced response rates in patients. One of the critical mechanisms of resistance to 5-FU appears to be the acute induction in thymidylate synthase (TS) levels following therapy with inhibitors of this enzyme. This mechanism is based on a novel autoregulatory feedback pathway wherein the TS protein regulates its own translational efficiency. Regulatory function of the enzyme is dependent on its state of occupancy by either the physiologic ligands or inhibitors, including fluoropyrimidines and antifolates. Ongoing efforts are directed toward utilizing knowledge of this protein/messenger RNA interaction for therapeutic benefit. Given the importance of TS, our laboratory has developed antibodies capable of quantitating the levels of this enzyme in fresh or paraffin-embedded tissues. Preliminary investigations suggest that the level of TS has prognostic importance in patients with rectal carcinoma and may be used to predict responsiveness to fluoropyrimidine agents. Novel strategies utilizing dual modulation of 5-FU with leucovorin and interferon are under investigation in both the advanced and adjuvant disease settings. Emerging mechanistic concepts regarding TS, along with the development of new, more potent inhibitors will hopefully result in future therapeutic gains.
Journal Article↗
Severe neurotoxicity following 5-fluorouracil-based chemotherapy in a patient with dihydropyrimidine dehydrogenase deficiency.
Patients with decreased dihydropyrimidine dehydrogenase (DPD) activity are at increased risk for experiencing serious adverse reactions following 5-fluorouracil (5-FU)-based chemotherapy. Symptoms include severe and potentially life-threatening gastrointestinal toxicity, myelosuppression, and neurological toxicity. In the present study, we describe a 50-year-old Caucasian man who developed severe encephalopathy during his second cycle of 5-FU chemotherapy. The patient remained in a comatose state for 4 days but then showed dramatic improvement in his neurological status following continuous i.v. infusion of thymidine at 8 g/m2/day. Laboratory studies revealed the patient to be severely DPD deficient, as demonstrated by DPD enzyme activity from peripheral blood mononuclear cells being below the lower limit of the 95th percentile of a control population and by Western immunoblot analysis showing undetectable levels of DPD protein. Additional studies revealed a significant defect in pyrimidine catabolism with a 3.3- and 365-fold increase in the levels of uracil in plasma and urine, respectively, compared to normal subjects. Family studies suggest that the inheritance pattern of this syndrome is complex and most consistent with an autosomal recessive trait. This study demonstrates that cancer patients with DPD deficiency are at increased risk for developing severe neurological toxicity secondary to 5-FU chemotherapy, and that infusional thymidine should be considered as a potential rescue agent against this particular host toxicity.
Brain Diseases↗
Thymidylate synthase gene amplification in human colon cancer cell lines resistant to 5-fluorouracil.
A series of 5-fluorouracil (5-FU)-resistant human colon H630 cancer cell lines were established by continuous exposure of cells to 5-FU. The concentration of 5-FU required to inhibit cell proliferation by 50% (IC50) in the parent colon line (H630) was 5.5 microM. The 5-FU IC50 values for the resistant H630-R1, H630-R10, and H630-R cell lines were 11-, 29-, and 27-fold higher than that for the parent H630 cell line. Using both the radioenzymatic 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) binding and catalytic assays for measurement of thymidylate synthase (TS) enzyme activity, there was significantly increased TS activity in resistant H630-R1 (13- and 23-fold), H630-R10 (37- and 40-fold), and H630-R (24- and 34-fold) lines, for binding and catalytic assays, respectively, compared with the parent H630 line. The level of TS protein, as determined by western immunoblot analysis, was increased markedly in resistant H630-R1 (23-fold), H630-R10 (33-fold), and H630-R (26-fold) cells. Northern analysis revealed elevations in TS mRNA levels in H630-R1 (18-fold), H630-R10 (39-fold), and H630-R (36-fold) cells relative to parent H630 cells. Although no major rearrangements of the TS gene were noted by Southern analysis, there was significant amplification of the TS gene in 5-FU-resistant cells, which was confirmed by DNA slot blot analysis. These studies demonstrate that continuous exposure of human colon cancer cells to 5-FU leads to TS gene amplification and overexpression of TS protein with resultant development of fluoropyrimidine resistance.
Cell Division↗
Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA.
Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism. In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA. Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807. Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM). A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements. E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA. These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains. Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation.
Base Sequence↗
Bone marrow aplasia and severe skin rash after a single low dose of methotrexate.
A 64 year old man with recurrent metastatic squamous cell carcinoma of the head and neck developed severe skin rash and bone marrow aplasia 4 and 7 days, respectively, following a single dose of 40 mg/m2 methotrexate (MTX). Skin rash involved regions of the face, lower abdomen, back, buttocks and both upper thighs. Biopsy of the skin rash demonstrated superficial perivascular lymphocytic infiltrate and was consistent with a drug reaction. Peripheral blood count revealed pancytopenia and a bone marrow biopsy was consistent with aplasia. Blood counts returned to normal 6 days after institution of granulocyte colony stimulating factor therapy. In the absence of mucositis or diarrhea, severe dermatologic toxicity following a single low dose of the drug suggests an 'allergic' or acute hypersensitivity reaction to MTX in this patient. Development of an extensive skin rash following a single dose of MTX may be an early warning sign for life-threatening bone marrow aplasia.
Carcinoma, Squamous Cell↗
Thymidylate synthase binds to c-myc RNA in human colon cancer cells and in vitro.
Using an immunoprecipitation-reverse transcription-PCR technique, we characterized a thymidylate synthase (TS) ribonucleoprotein complex in cultured human colon cancer cells that consists of TS protein and the mRNA of the nuclear oncogene c-myc. TS protein is complexed in intact cells with the C-terminal coding region of c-myc mRNA that includes nucleotide positions 1625 to 1790. RNA electrophoretic gel mobility shift assays confirm a specific interaction between TS protein and c-myc mRNA and provide additional evidence that the C-terminal coding region represents an important cis-acting regulatory element. Further evidence demonstrates that the in vitro translational efficiency of c-myc mRNA is inhibited as a result of its direct interaction with TS protein. In addition, the presence of exogenous c-myc mRNA specifically relieves the inhibitory effects of TS protein on TS mRNA translation.
Base Sequence↗
Intracardiac echocardiography during radiofrequency catheter ablation of cardiac arrhythmias in humans.
OBJECTIVES: The purpose of this study was to describe our preliminary experience using catheter-based intracardiac echocardiography as an adjunct to biplane fluoroscopy for guiding radiofrequency catheter ablation of atrial arrhythmias in the right side of the heart. BACKGROUND: Catheter ablation requires precise positioning and stable ablation electrode-endocardial contact. This procedure is currently guided by an analysis of intracardiac electrograms and fluoroscopy. However, the use of fluoroscopy does not allow the endocardium and certain anatomic landmarks to be identified and is associated with the hazards of radiation exposure. METHODS: Seventeen symptomatic patients were studied. A 10F 10-MHz intracardiac imaging catheter was used to visualize specific anatomic landmarks in the right atrium for directing the ablation electrode in 15 patients undergoing radiofrequency ablation of 19 arrhythmias and to assist with interatrial septal puncture in 3 patients. RESULTS: Continuous intracardiac imaging was performed for a mean +/- SD of 63.6 +/- 39.2 min and demonstrated distal electrode-endocardial tissue contact in 81 (60%) of 134 radiofrequency applications. Movement of the catheter was demonstrated during 36 (44%), microcavitations during 39 (48%) and thrombus during 15 (19%) of the 81 imaged applications. In 7 of 10 procedures for atrial flutter, successful ablation was directed at anatomic corridors in the right atrium visualized with intracardiac echocardiography. During ablation of atrial tachycardia, imaging identified abnormal atrial anatomy related to previous surgery and guided successful ablation of a reentrant tachycardia circulating around these anatomic obstacles. In two procedures for slow pathway modification of atrioventricular node reentrant tachycardia, intracardiac echocardiography confirmed catheter stability at the tricuspid annulus anterior to the coronary sinus. CONCLUSIONS: During catheter ablation, intracardiac echocardiography augments fluoroscopy by visualizing anatomic landmarks, ensuring stable endocardial contact and assisting in transseptal puncture. Ablation of typical atrial flutter can be successfully directed at anatomic corridors identified using intracardiac imaging.
Atrial Flutter↗
The effect of reducing reagents on binding of thymidylate synthase protein to thymidylate synthase messenger RNA.
Human thymidylate synthase (TS) protein specifically binds to its own TS mRNA and functions as a translational repressor. In the presence of reducing agents, the RNA binding activity of TS protein is significantly enhanced. In contrast, treatment of TS protein with the oxidizing agent diamide inhibits RNA binding. Scatchard analysis reveals that in the presence of the reducing agent 2-mercaptoethanol, the TS protein/TS mRNA interaction changes from low (Kd = 66 nM) to high (Kd = 2.6 nM) apparent affinity. The catalytic activity of TS is increased by up to 6.5-fold in the presence of 2-mercaptoethanol. These studies demonstrate that the interaction between TS protein and its target TS mRNA is sensitive to the presence of reducing reagents and is dependent upon a reversible sulfhydryl switch mechanism.
Binding Sites↗
Interaction of Pneumocystis carinii dihydropteroate synthase with sulfonamides and diaminodiphenyl sulfone (dapsone).
Dihydropteroate synthase is the target enzyme for the sulfonamide compounds, which are the mainstay of therapy for Pneumocystis carinii pneumonia, a common infection in patients with impaired immunity. The stability of this enzyme, its kinetic constants with respect to substrates, and the 50% inhibitory concentration (IC50) of several sulfonamides and the sulfone dapsone have been characterized using both cell-free and intact organism assay systems. Stability of the enzyme is dependent on storage temperature, reducing reagents, and to a lesser extent, protease inhibitors. The sulfonamides sulfadiazine and sulfamethoxazole were found to be highly potent inhibitors of P. carinii dihydropteroate synthase with IC50s of 0.42 and 0.71 microM, respectively. Dapsone had equivalent potency when compared with the most potent sulfonamides tested in both assay systems. Data suggest that sulfamethoxazole, sulfadiazine and dapsone may represent equivalent choices as P. carinii dihydropteroate synthase inhibitors, assuming an equivalent in vivo drug exposure can be achieved.
4-Aminobenzoic Acid↗
Identification of a thymidylate synthase ribonucleoprotein complex in human colon cancer cells.
Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.
Base Sequence↗
Radiofrequency catheter ablation guided by intracardiac echocardiography.
BACKGROUND: Radiofrequency catheter ablation requires precise positioning of the ablation electrode. Fluoroscopically guided catheter manipulation has limitations, and there are risks of radiation exposure. The purpose of this study was to examine the feasibility of guiding catheter ablation within the right atrium with catheter-based intracardiac echocardiography. METHODS AND RESULTS: A 10F, 10-MHz intracardiac imaging catheter was used to direct an ablation electrode at four or five anatomic landmarks in the right atrium. Thirty-eight radiofrequency energy applications were performed in nine anesthetized dogs, and 38 lesions were identified on pathological examination. Lesions were created a mean of 1.9 +/- 2.1 mm from the ultrasound-guided site. Twenty-six of 38 lesions (68%) were less than 2.2 mm from the imaged site. Intracardiac echocardiography also was used to confirm stable electrode-endocardial contact in 37 energy applications (97%) and identified catheter movement in 9 energy applications (24%). Discrete lesions, microcavitations, and thrombi were observed in 13 (34%), 23 (61%), and 19 (50%) of 38 energy applications, respectively. Microcavitations predicted the appearance of thrombus. Fluoroscopy time required to create four or five lesions decreased from 23 minutes in the first study to less than 2 minutes in the last five studies. CONCLUSIONS: Catheter-based intracardiac echocardiography can accurately guide catheter ablation directed at anatomic landmarks and potentially reduced ionizing radiation exposure. Intracardiac imaging can be used to confirm endocardial contact, identify electrode movement, and directly visualize lesions. Intracardiac echocardiography also can be used to identify microcavitations, which predict thrombus formation during radiofrequency energy applications.
Animals↗
Effects of 5-fluorouracil substitution on the RNA conformation and in vitro translation of thymidylate synthase messenger RNA.
In vitro transcribed thymidylate synthase (TS) mRNA which is 100% substituted with 5-fluorouracil (FUra) was analyzed for changes in mRNA secondary structure, for alterations in translational efficiency, and for evidence of translational miscoding in vitro. FUra substitution in TS mRNA results in an altered migration pattern in non-denaturing RNA gels and in decreased hyperchromicity in RNA melting temperature studies, consistent with a change in mRNA secondary structure. However, no change in the translational efficiency of FUra-substituted TS mRNA is seen compared to control TS mRNA in either rabbit reticulocyte lysate or wheat germ extract in vitro translation systems. Analysis of the in vitro translation product of FUra-substituted TS mRNA by Western immunoblotting, isoelectric focusing, 5-fluoro-2'-deoxyuridine 5'-monophosphate binding, and TS catalytic activity experiments shows no difference compared to control TS mRNA. We conclude that the in vitro translation products of FUra-substituted and control TS mRNA are identical. Our findings do not support the hypothesis that changes in the mRNA template are responsible for the RNA-directed cytotoxicity of FUra.
Blotting, Western↗
Sensitivity of tumoricidal function in macrophages from different anatomical sites of cancer patients to modulation of arachidonic acid metabolism.
The sensitivity of cancer patient macrophages from different anatomical sites to arachidonic acid metabolism was investigated in tumor cell cytotoxicity assays. Alveolar macrophages and peripheral blood monocytes from 13 non-small cell lung cancer patients, peritoneal macrophages and peripheral blood monocytes from 13 ovarian cancer patients, and comparable macrophages from control patients with nonmalignant lung or gynecological diseases were tested. Inhibitors of either the cyclooxygenase pathway or the lipoxygenase pathway together with specific metabolites of each pathway were used to evaluate how these different macrophage populations are regulated by eicosanoids. In addition, metabolic studies were performed to compare directly the arachidonic acid metabolism of macrophages obtained from these different anatomical locations. The results demonstrate that the peripheral blood monocytes from lung cancer and ovarian cancer patients and the peritoneal macrophages from ovarian cancer patients are sensitive to cyclooxygenase inhibition; this was not seen with comparable macrophages from the relevant control patients. Sensitivity to modulation by cyclooxygenase inhibition correlated with increased cyclooxygenase metabolism and with the capacity of prostaglandin to mediate suppression of tumoricidal function in these populations of cancer patient macrophages. In contrast, alveolar macrophages from cancer patients were not sensitive to either cyclooxygenase inhibition or to prostaglandin-mediated suppression. No such differential influences were revealed for the lipoxygenase pathway of arachidonic acid metabolism in any macrophage population tested. Thus, eicosanoids, particularly those of the cyclooxygenase pathway, can be a critical immunoregulatory feature of certain tumor microenvironments.
6-Ketoprostaglandin F1 alpha↗
Specific binding of human dihydrofolate reductase protein to dihydrofolate reductase messenger RNA in vitro.
Dihydrofolate reductase (DHFR) is a critical enzyme in de novo purine and thymidylate biosynthesis. An RNA gel mobility shift assay was used to demonstrate a specific interaction between human recombinant DHFR protein and its corresponding DHFR mRNA. Incubation of DHFR protein with either its substrates, dihydrofolate or NADPH, or with an inhibitor, methotrexate, repressed its ability to interact with DHFR mRNA. An in vitro rabbit reticulocyte lysate translation system was used to show that the addition of exogenous human recombinant DHFR protein to in vitro translation reactions specifically inhibited DHFR mRNA translation. These studies suggest that the direct interaction between DHFR protein and its mRNA may be a mechanism for regulation of DHFR synthesis.
Base Sequence↗