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Biomedical subjects

Edward Chu

Publications and source records attributed to Edward Chu.

At least 37 records · Page 2Linked to original sources
PubMed2005

Current therapies for advanced colorectal cancer.

Significant advances have been made in the treatment of advanced colorectal cancer over the past 5 years, namely due to the introduction of three novel cytotoxic agents-capecitabine (Xeloda), irinotecan (Camptosar), and oxaliplatin (Eloxatin)-and the recent approval of two biologic agents-bevacizumab (Avastin) and cetuximab (Erbitux). During this time period, the median survival of patients with advanced, metastatic disease has gone from 10 to 12 months to nearly 24 months. Intense efforts have focused on identifying novel targeted therapies that target specific growth factor receptors, critical signal transduction pathways, and/or key pathways that mediate the process of angiogenesis. Recent clinical trial results suggest that the anti-VEGF antibody bevacizumab can be safely and effectively used in combination with each of the active anticancer agents used in colorectal cancer. Despite the development of active combination regimens, significant improvements in the actual cure rate have not yet been achieved. Combination regimens with activity in advanced disease are being evaluated in the adjuvant and neoadjuvant settings. The goal is to integrate these targeted strategies into standard chemotherapy regimens so as to advance the therapeutic options for the treatment of advanced colorectal cancer. Finally, intense efforts are attempting to identify the critical molecular biomarkers that can be used to predict for either clinical response to chemotherapy and/or targeted therapies and/or the drug-specific side effects. The goal of such studies is to facilitate the evolution of empiric chemotherapy to individually tailored treatments for patients with colorectal cancer.

Antibodies, Monoclonal↗
PubMed2004

Translational autoregulation of thymidylate synthase and dihydrofolate reductase.

The folate-dependent enzymes, thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are critical for providing the requisite nucleotide precursors for maintaining DNA synthesis and DNA repair. In addition to their essential roles in enzyme catalysis, these two enzymes have now been shown to function as RNA binding proteins. Using in vitro and in vivo experimental model systems, we have shown that the functional consequence of binding of TS protein to its own cognate mRNA, as well as binding of DHFR to its own DHFR mRNA, is translational repression. Herein, we review and update studies focusing on the translational autoregulatory control of TS and DHFR expression and discuss the molecular elements that are required for these specific RNA-protein interactions. Moreover, we present evidence showing that abrogation of these normal translational autoregulatory feedback mechanisms provides the molecular basis for the rapid development of cellular drug resistance.

Animals↗
PubMed2004

Characterization of a cis-acting regulatory element in the protein-coding region of human dihydrofolate reductase mRNA.

Previous studies have shown that human DHFR (dihydrofolate reductase), in addition to its critical role in DNA biosynthesis, functions as an RNA-binding protein. The interaction between DHFR and its own mRNA results in translational repression. In this study, we characterized the cis-acting elements on human DHFR mRNA that are required for the DHFR mRNA-DHFR protein interaction. Using a series of gel-shift and nitrocellulose filter-binding assays, a 164 nt RNA sequence, corresponding to nt 401-564, was identified within the coding region that binds to DHFR protein with an affinity similar to that of full-length DHFR mRNA. To document in vivo biological activity, various DHFR sequences contained within the coding region were cloned on to the 5' end of a luciferase reporter plasmid, and transient transfection experiments were performed using human colon cancer RKO cells. In cells transfected with p644/DHFR:401-564, luciferase activity was decreased by 50% when compared with cells transfected with the p644 plasmid alone. Luciferase mRNA levels were identical under each of these conditions, as determined by Northern-blot analysis. In cells transfected with p644/DHFR:401-564, luciferase activity was restored to almost 100% of control when cells were treated with the antifolate analogue methotrexate or with a short-interfering RNA targeting DHFR mRNA. These findings provide evidence that the DHFR 401-564 sequence is a DHFR-response element. In vitro and in vivo studies further localized this cis-element to an 82 nt sequence corresponding to nt 401-482. This work provides new insights into critical elements that mediate RNA-protein interactions.

Binding Sites↗
PubMed2004

Small interfering double-stranded RNAs as therapeutic molecules to restore chemosensitivity to thymidylate synthase inhibitor compounds.

RNA interference is a post-transcriptional mechanism by which double-stranded RNA specifically silence expression of a corresponding gene. Small interfering double-stranded RNA (siRNA) of 21-23 nucleotides can induce the process of RNA interference. Studies from our laboratory have shown that translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this process gives rise to increased synthesis of TS and leads to the development of cellular drug resistance to TS-targeted compounds. As a strategy to inhibit TS expression at the mRNA level, siRNAs were designed to target nucleotides 1058-1077 on human TS mRNA. Transfection of TS1058 siRNA into human colon cancer RKO cells resulted in a dose-dependent inhibition of TS expression with an IC(50) value of 10 pM but had no effect on the expression of alpha-tubulin or topoisomerase I. Inhibition of TS expression by TS1058 was maximal at 48 h and remained suppressed for up to 5 days. Pretreatment of RKO cells with TS1058 siRNA suppressed TS protein induction following exposure to raltitrexed. In addition, TS1058 restored chemosensitivity of the resistant RKO-HTStet cell line to various TS inhibitor compounds. On treatment with TS1058, IC(50) values for raltitrexed, 1843U89, and 5-fluoro-2'-deoxyuridine decreased by approximately 15-16-fold. These studies suggest that TS-targeted siRNAs are effective inhibitors of TS expression and may have therapeutic potential by themselves or as chemosensitizers in combination with TS inhibitor compounds.

Cell Division↗
PubMed2003

Role of cysteine amino acid residues on the RNA binding activity of human thymidylate synthase.

The role of cysteine sulfhydryl residues on the RNA binding activity of human thymidylate synthase (TS) was investigated by mutating each cysteine residue on human TS to a corresponding alanine residue. Enzymatic activities of TS:C43A and TS:C210A mutant proteins were nearly identical to wild-type TS, while TS:C180A and TS:C199A mutants expressed >80% of wild-type enzyme activity. In contrast, TS:C195A was completely inactive. Mutant proteins, TS:C195A, TS:C199A and TS:C210A, retained RNA binding activity to nearly the same degree as wild-type human TS. RNA binding activity of TS:C43A was reduced by 30% when compared to wild-type TS, while TS:C180A was completely devoid of RNA binding activity. In vitro translation studies confirmed that mutant proteins TS:C43A, TS:C195A, TS:C199A and TS:C210A, significantly repressed human TS mRNA translation, while TS:C180A was unable to do so. To confirm the in vivo significance of the cysteine sulfhydryl residue, mutant proteins TS:C180A and TS:C195A were each expressed in human colon cancer HCT-C18:TS(-) cells that expressed a functionally inactive TS. A recombinant luciferase reporter gene under the control of a TS-response element was co-transfected into these same cells, and luciferase activity increased in the presence of the TS:C195A mutant TS protein to a level similar to that observed upon expression of wild-type TS protein. In contrast, luciferase activity remained unchanged in cells expressing the TS:C180A mutant protein. Taken together, these findings identify Cys-180 as a critical residue for the in vitro and in vivo translational regulatory effects of human TS.

Alanine↗
PubMed2003

Thymidylate synthase inhibitors as anticancer agents: from bench to bedside.

Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate to 2'-deoxythymidine-5'-monophosphate. This pathway provides the sole intracellular de novo source of 2'-deoxythymidine-5'-triphosphate; therefore, TS represents a critical target in cancer chemotherapy. 5-Fluorouracil (5-FU) was synthesized in 1957 and represents the first class of antineoplastic agents to be developed as inhibitors of TS. While 5-FU has been widely used to treat various human malignancies, its overall clinical efficacy is limited. Therefore, significant efforts have focused on the design of novel, more potent inhibitor compounds of TS. These agents fall into two main categories: folate analogs and nucleotide analogs. Five antifolate analogs are currently being evaluated in the clinic: raltitrexed, pemetrexed, nolatrexed, ZD9331, and GS7904L. Our laboratory has identified a novel mechanism of resistance that develops to TS inhibitor compounds, namely drug-mediated acute induction of new TS synthesis; this mechanism is directly controlled at the translational level. The ability of cancer cells to acutely induce the expression of TS may represent a novel mechanism for the development of cellular drug resistance. The future success of TS inhibitor compounds in the clinic may depend on novel strategies to selectively inhibit TS and on novel combination therapies to overcome cellular drug resistance.

Antineoplastic Agents↗
PubMed2002

Identification of critical amino acid residues on human dihydrofolate reductase protein that mediate RNA recognition.

Previous studies have shown that human dihydrofolate reductase (DHFR) acts as an RNA-binding protein, in which it binds to its own mRNA and, in so doing, results in translational repression. In this study, we used RNA gel mobility shift and nitrocellulose filter-binding assays to further investigate the specificity of the interaction between human DHFR protein and human DHFR mRNA. Site-directed mutagenesis was used to identify the critical amino acid residues on DHFR protein required for RNA recognition. Human His-Tag DHFR protein specifically binds to human DHFR mRNA, while unrelated proteins including thymidylate synthase, p53 and glutathione-S-transferase were unable to form a ribonucleoprotein complex with DHFR mRNA. The Cys6 residue is essential for RNA recognition, as mutation at this amino acid with either an alanine (C6A) or serine (C6S) residue almost completely abrogated RNA-binding activity. Neither one of the cysteine mutant proteins was able to repress the in vitro translation of human DHFR mRNA. Mutations at amino acids Ile7, Arg28 and Phe34, significantly reduced RNA-binding activity. An RNA footprinting analysis identified three different RNA sequences, bound to DHFR protein, ranging in size from 16 to 45 nt, while a UV cross-linking analysis isolated an approximately 16 nt RNA sequence bound to DHFR. These studies begin to identify the critical amino acid residues on human DHFR that mediate RNA binding either through forming direct contact points with RNA or through maintaining the protein in an optimal structure that allows for the critical RNA-binding domain to be accessible.

Amino Acids↗
PubMed2002

Thymidylate synthase as a translational regulator of cellular gene expression.

Studies from our laboratory have shown that the folate-dependent enzyme, thymidylate synthase (TS), functions as an RNA binding protein. There is evidence that TS, in addition to interacting with its own TS mRNA, forms a ribonucleoprotein complex with a number of other cellular mRNAs, including those corresponding to the p53 tumor suppressor gene and the myc family of transcription factors. Using both in vitro and in vivo model systems, we have demonstrated that the functional consequence of binding of TS protein to its own cognate mRNA, as well as binding of TS to the p53 mRNA, is translational repression. Herein, we review current work on the translational autoregulatory control of TS expression and discuss the molecular elements that are required for the TS protein-TS mRNA interaction. TS may play a critical role in regulating the cell cycle and the process of apoptosis through its regulatory effects on expression of p53 and perhaps other cell cycle related proteins. Finally, the ability of TS to function as a translational regulator may have important consequences with regard to the development of cellular resistance to various anticancer drugs.

Animals↗