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D L Sackett

Publications and source records attributed to D L Sackett.

At least 55 records · Page 3Linked to original sources
PubMed1997

Paclitaxel-resistant human ovarian cancer cells have mutant beta-tubulins that exhibit impaired paclitaxel-driven polymerization.

Acquired resistance to paclitaxel can be mediated by P-glycoprotein or by alterations involving tubulin. We report two paclitaxel-resistant sublines derived from 1A9 human ovarian carcinoma cells. Single-step paclitaxel selection with verapamil yielded two clones that are resistant to paclitaxel and collaterally sensitive to vinblastine. The resistant sublines are not paclitaxel-dependent, and resistance remained stable after 3 years of drug-free culture. All cell lines accumulate [3H]paclitaxel equally, and no MDR-1 mRNA was detected by polymerase chain reaction following reverse transcription. Total tubulin content is similar, but the polymerized fraction increased in parental but not in resistant cells following the paclitaxel addition. Purified tubulin from parental cells demonstrated paclitaxel-driven increased polymerization, in contrast to resistant cell tubulin, which did not polymerize under identical conditions. In contrast, epothilone B, an agent to which the resistant cells retained sensitivity, increased assembly. Comparable expression of beta-tubulin isotypes was found in parental and resistant cells, with predominant expression of the M40 and beta2 isotypes. Sequence analysis demonstrated acquired mutations in the M40 isotype at nucleotide 810 (T --> G; Phe270 --> Val) in 1A9PTX10 cells and nucleotide 1092 (G --> A; Ala364 --> Thr) in 1A9PTX22 cells. These results identify residues beta270 and beta364 as important modulators of paclitaxel's interaction with tubulin.

Antineoplastic Agents, Phytogenic↗
PubMed1997

Tubulin from paclitaxel-resistant cells as a probe for novel antimicrotubule agents.

PURPOSE: Treatment with paclitaxel (PTX) can lead to the appearance of drug resistance with accompanying changes in tubulin. The purpose of this study was to develop an assay for microtubule-active agents that are able to circumvent changes in tubulin that result in acquired resistance to paclitaxel. METHODS: The assay measured the promotion of microtubule polymerization when target agents were added to solutions containing tubulin purified from cultured cells. Tubulin was prepared from PTX-sensitive 1A9 ovarian carcinoma cells and from a PTX-resistant clone. Polymerization was monitored spectrophotometrically and validated by electron microscopy. RESULTS: Exposure of tubulin isolated from PTX-sensitive 1A9 ovarian carcinoma cells to substoichiometric PTX resulted in polymerization equivalent to that observed with brain tubulin. In contrast, tubulin from a PTX-resistant 1A9 clone failed to polymerize under identical conditions. If a C-2-modified analog of PTX (2-debenzoyl-2-(m-azidobenzoyl)paclitaxel) was substituted for PTX in the same experiment, the tubulins from both sensitive and resistant cells polymerized as well as brain tubulin. As predicted from these results, the PTX analog was nearly as cytotoxic to the PTX-resistant cells as it was to the parental cells: the relative resistance of the resistant cells compared to the parental is only 3-5-fold for the PTX analog versus 25-30-fold for PTX. CONCLUSION: Polymerization of purified tubulin from the paclitaxel-resistant cells provided an assay for agents able to circumvent the tubulin alterations that result in acquired paclitaxel resistance.

Animals↗
PubMed1997

Evidence-based medicine.

Evidence-based medicine, whose philosophical origins extend back to mid-19th century Paris and earlier, is the conscientious, explicit and judicious use of current best evidence in making decisions about the care of individual patients. The practice of evidence-based medicine means integrating individual clinical expertise with the best available external clinical evidence from systematic research. By individual clinical expertise we mean the proficiency and judgment that we individual clinicians acquire through clinical experience and clinical practice. Increased expertise is reflected in many ways, but especially in more effective and efficient diagnosis and in the more thoughtful identification and compassionate use of individual patients' predicaments, rights, and preferences in making clinical decisions about their care. By best available external clinical evidence we mean clinically relevant research, often from the basic sciences of medicine, but especially from patient centered clinical research into the accuracy and precision of diagnostic tests (including the clinical examination), the power of prognostic markers, and the efficacy and safety of therapeutic, rehabilitative, and preventive regimens. External clinical evidence both invalidates previously accepted diagnostic tests and treatment and replaces them with new ones that are more powerful, more accurate, more efficacious, and safer. Good doctors use both individual clinical expertise and the best available external evidence, and neither alone is enough. Without clinical expertise, practice risks becoming tyrannized by external evidence, for even excellent external evidence may be inapplicable to or inappropriate for an individual patient. Without current best external evidence, practice risks becoming rapidly out of date, to the detriment of patients. The practice of evidence-based medicine is a process of life-long, self-directed learning in which caring for our own patients creates the need for clinically important information about diagnosis, prognosis, therapy, and other clinical and health care issues, and in which we (1) convert these information needs into answerable questions; (2) track down, with maximum efficiency, the best evidence with which to answer them (whether from the clinical examination, the diagnostic laboratory from research evidence, or other sources); (3) critically appraise that evidence for its validity (closeness to the truth) and usefulness (clinical applicability); (4) integrate this appraisal with our clinical expertise and apply it in practice; and (5) evaluate our performance.

Evidence-Based Medicine↗
PubMed1997

Natural osmolyte trimethylamine N-oxide stimulates tubulin polymerization and reverses urea inhibition.

The natural osmolyte trimethylamine N-oxide (TMAO) is one of the methylamine compounds often accumulated by diverse organisms in response to osmotic stress and/or to compensate for the deleterious effects of urea. Tubulin polymerization is promoted by TMAO. At 1 M TMAO, tubulin polymers are produced with properties expected of normal steady-state microtubules (MT): polymerization is reversed by exposure to cold or the antimitotic drug podophyllotoxin, a critical concentration for polymerization at 30 degrees C of 1.5 microM is found, and the morphology of the polymers in electron micrographs is typical of MT and ribbons, or open MT. At 2 M TMAO, polymerization is very rapid and hyperstable polymers are formed. These are resistant to cold-induced depolymerization although still sensitive to podophyllotoxin inhibition. A lower critical concentration of 0.7 microM is observed, and electron micrographs reveal MT, ribbons, and other polymer forms not usually stable, such as splayed protofilaments. Inhibition of tubulin polymerization by low concentrations of urea (Sackett, D. L., B. Bhattacharyya, and J. wolff. Biochemistry 33: 12868-12878, 1994) is largely reversed by the presence of TMAO at one-half the molarity of urea, the physiological ratio observed in cartilaginous fishes. Other methylamines, including betaine, dimethylglycine, glycine, and sarcosine, failed to stimulate MT polymerization or protect against urea inhibition. Trimethylamine, taurine and glycylglycine inhibit polymerization. TMAO did not interfere with binding of MT-associated proteins (MAP) and protected both tubulin assembly and MAP binding from urea.

Animals↗
PubMed1996

Interaction of a fluorescent derivative of paclitaxel (Taxol) with microtubules and tubulin-colchicine.

A fluorescent derivative of paclitaxel, 2-debenzoyl-2-(m-aminobenzoyl)paclitaxel (2-AB-PT), has been prepared. 2-AB-PT induces microtubule assembly in vitro, but is about 3-fold less potent than paclitaxel itself. The absorption and emission characteristics of 2-AB-PT were analyzed as a function of solvent. It was found that both spectra were perturbed by specific solvent effects when the solvent contained a hydrogen bond donor. The absorption and fluorescence spectra of 2-AB-PT bound to microtubules could not be mimicked by a single solvent, but the absorption and emission maxima of the tubulin-bound species could be duplicated by a solvent mixture of DMSO and water. These results indicate that the fluorophore binding site on the microtubule is in an environment of intermediate polarity that is accessible to a hydrogen bond donor in the vicinity of the m-amino group. In addition, tubulin fluorescence is quenched in the 2-AB-PT/microtubule complex, and energy transfer from tubulin to 2-AB-PT is apparent. These results indicate that substituents on the C-2 position of paclitaxel associate with tubulin when bound to the microtubule. 2-AB-PT binding to microtubules was quantitatively analyzed by fluorescence titrations. Two classes of binding sites for 2-AB-PT on microtubules were found. The high affinity site has an apparent association constant (K1app) of 2.0 (+/- 0.9) x 10(7) M-1 and an apparent binding stoichiometry (n1app) of 0.8 (+/- 0.1) sites/tubulin dimer in the microtubule. The apparent association constant for the lower affinity site is about 100-fold less than that of the higher affinity site (K2app = 2.1 (+/- 0.7) x 10(5) M-1), and the stoichiometry of the lower affinity site or class of sites (n2app) was found to be 1.3 +/- 0.1. Paclitaxel blocked 2-AB-PT binding to the high affinity site. No binding of 2-AB-PT to unassembled tubulin was observed, but the emission spectrum of 2-AB-PT in the presence of the tubulin-colchicine complex resembled the emission spectrum of the ligand bound to microtubules. It was previously shown that paclitaxel can induce GTPase activity in the tubulin-colchicine complex, indicating that paclitaxel can bind to unassembled tubulin in its complex with colchicine [Carlier, M.-F., & Pantaloni, D. (1983) Biochemistry 22, 4814-4822]. Rigorous characterization of the aggregation state of the protein under these conditions demonstrates that 2-AB-PT is also capable of binding to the tubulin-colchicine complex.

Animals↗
PubMed1996

The multiple origins of cooperativity in binding to multi-site lattices.

Binding events involving the reversible association of ligands with polymeric lattices of binding sites are common in biology and frequently exhibit significant cooperativity in binding. Positive and negative cooperativity in binding may be detected by characteristic changes in binding curves for multiple binding, compared to the binding expected for simple, independent binding events that are based on combinatorial considerations only. Cooperativity arises from ligand-dependent interactions distinct from binding per se. Ligand-dependent nearest neighbor interactions may be of two types referred to as ligand-lattice (which can only occur if a bound ligand is unneighbored) and ligand-ligand (which can occur if two or more bound ligands are adjacent). The molecular mechanisms underlying these two sources of cooperativity are not the same. Identical cooperative binding curves may be produced by changes from unity in parameters representing either one or both of these interaction types. Positive cooperativity may equally result from destabilizing ligand-lattice interactions that disfavor initial, unneighbored binding, stabilizing ligand-ligand interactions that favor subsequent, neighbored binding, or both. The structural origins of these are different, and cooperativity may emerge from multiple structural interactions.

Binding Sites↗
PubMed1996

Outcome of patients discharged from a coronary care unit with a diagnosis of "chest pain not yet diagnosed".

OBJECTIVE: To determine the outcome and 3-year mortality rate among patients discharged from a coronary care unit (CCU) with a diagnosis of "chest pain not yet diagnosed." DESIGN: Prospective observational cohort study. SETTING: CCU in a university teaching hospital. PATIENTS: All 158 eligible patients discharged from the CCU between August 1986 and December 1988. Of them, 27 refused to participate and 31 did not meet the inclusion criteria because of significant co-morbidity or transportation difficulties. INTERVENTIONS: Evaluation with maximal and thallium exercise stress testing and four major gastrointestinal (GI) investigations: 24-hour intraesophageal pH monitoring, upper GI endoscopy with biopsy, esophageal motility studies and an upper GI barium series. OUTCOME MEASURES: Results of investigations and incidence of recurrent chest pain, CCU readmission, coronary angiography, coronary artery bypass surgery, myocardial infarction and death at 6, 12, 24 and 36 months after the index visit. RESULTS: Of the patients enrolled in the study 79% (79/100) had a normal exercise thallium stress test result, 74% (68/92) had an abnormal result from the 24-hour pH monitoring, 87% (82/94) had abnormal endoscopic results, 90% (84/93) had abnormal manometric results, and 89% (83/93) had signs of reflux with the barium series. At 3 years 50 patients had recurrent chest pain and 3 underwent coronary artery bypass surgery. Three patients died over the 3 years, all of noncardiac causes. CONCLUSION: Many patients discharged from the CCU with a diagnosis of chest pain not yet diagnosed have a high incidence of esophageal disorders and a very low 3-year mortality rate. More research into the early and effective identification and management of patients with such a diagnosis is needed.

Adult↗
PubMed1996

Charge-shielding and the "paradoxical" stimulation of tubulin polymerization by guanidine hydrochloride.

Low concentrations of guanidine hydrochloride (GuHCl) increase the rate (and to a lesser degree, the extent) of tubulin polymerization as assessed by light scattering. Maximum enhancement occurs at 120-160 mM GuHCl followed by decreases at higher GuHCl. The latent period is decreased, and there is a 3-4 fold reduction in the critical concentration of polymerization. Electronmicrographs reveal microtubules in the controls and an increasing fraction of total polymers present as aberrant microtubules as the GuHCl concentration is increased from 20 to 100 mM. The GuHCl effect is markedly reduced, but not abolished, in tubulin S (in which the anionic C termini of both monomers have been removed). The GuHCl-induced polymerization has an absolute requirement for GTP and taxol or DMSO, is very sensitive to podophyllotoxin inhibition, and can overcome urea-mediated inhibition of polymerization. Guanidinium analogues mimic the GuHCl effect roughly as a function of the number of potential hydrogen bonds. The anions of the guanidine salts superimpose their inhibitory action on the guanidinium cation effect according to the lyotropic series. At higher GuHCl concentrations (peak effect 500-700 mM), a different polymer (type II) is formed that is GTP and taxol independent, but whose polymerization is retarded but not prevented by podophyllotoxin. Its structure resembles the fibrillar network seen in unfolding intermediates of other proteins. We conclude that both charge and hydrogen-bonding ability are major contributors to the GuHCl-induced promotion of tubulin polymerization, and that charge-shielding is likely to be the basis for this effect.

Animals↗