Biomedical subjects

Nicole M Verrills

Publications and source records attributed to Nicole M Verrills.

10 recordsLinked to original sources

PubMed2006

Alterations in gamma-actin and tubulin-targeted drug resistance in childhood leukemia.

BACKGROUND: Proteomic investigations have revealed alterations in cytoskeletal proteins expressed in human acute lymphoblastic leukemia cells that are resistant to microtubule-disrupting agents. We characterized gamma-actin expression in antimicrotubule drug-resistant leukemia and examined the effect of altered gamma-actin in resistance of acute lymphoblastic leukemia to antimicrotubule agents. METHODS: Two-dimensional polyacrylamide gel electrophoresis and mass spectrometry were used to identify actin proteins in human acute lymphoblastic leukemia cell lines resistant to vinblastine (CCRF-CEM/VLB100 cells) and desoxyepothilone B (CCRF-CEM/dEpoB140 cells). Fluorescence-based cycle sequencing was used to detect gene mutations. Site-directed mutagenesis was used to generate mutant gamma-actin expression plasmids, which were used to transfect mouse NIH/3T3 cells. Clonogenic analysis was used for drug sensitivity studies. A small interfering RNA (siRNA) was used to block gamma-actin gene expression in human neuroblastoma SH-EP cells. Expression of gamma-actin (normalized to that of beta2-microglobulin [beta2M]) in primary leukemia cells obtained from patients at diagnosis (n = 44) and relapse (n = 25) was examined using semiquantitative reverse transcription-polymerase chain reaction. Statistical significance of changes in the ratio of gamma-actin to beta2M expression between diagnosis and relapse samples was determined by two-sided unpaired Student's t tests. RESULTS: We identified novel mutant forms of gamma-actin and the concomitant loss of wild-type gamma-actin in CCRF-CEM/VLB100 cells and CCRF-CEM/dEpoB140 cells. Mouse NIH/3T3 cells that expressed the mutant gamma-actin proteins were more resistant to antimicrotubule agents than cells transfected with empty plasmid. Human neuroblastoma SH-EP cells transfected with gamma-actin siRNA displayed higher relative resistance to paclitaxel (P<.001), vinblastine (P = .04), and epothilone B (P = .045) than mock-transfected cells. No gamma-actin gene mutations were identified in 37 samples of primary leukemia cells (eight from patients at diagnosis, 29 from patients at relapse). Gamma-actin gene expression was lower in acute lymphoblastic leukemia samples collected at clinical relapse (n = 25; mean gamma-actin/beta2M = 0.53) than in samples collected at diagnosis (n = 44; mean gamma-actin/beta2M = 0.68; difference = 0.15, 95% confidence interval [CI] = 0.04 to 0.27, P = .01). CONCLUSIONS: These data provide functional and associative clinical evidence of a novel form of drug resistance that involves interactions between gamma-actin and microtubules.

Actins↗

PubMed2006

Mast cell function: regulation of degranulation by serine/threonine phosphatases.

Mast cells play both effector and modulatory roles in a range of allergic and immune responses. The principal function of these cells is the release of inflammatory mediators from mast cells by degranulation, which involves a complex interplay of signalling molecules. Understanding the molecular architecture underlying mast cell signalling has attracted renewed interest as the capacity for therapeutic intervention through controlling mast cell degranulation is now accepted as a viable proposition. The dynamic regulation of signalling by protein phosphorylation is a well-established phenomenon and many of the early events involved in mast cell activation are well understood. Less well understood however are the events further downstream of receptor activation that allow movement of granules through the cytoskeletal barrier and docking and fusion of granules with the plasma membrane. Whilst a potential role for the protein phosphatase family of signalling enzymes in mast cell function has been accepted for some time, the evidence has largely been derived from the use of broad specificity pharmacological inhibitors and results often depend upon the experimental conditions, leading to conflicting views. In this review, we present and discuss the pharmacological and recent molecular evidence that protein phosphatases, and in particular the protein phosphatase serine/threonine phosphatase type 2A (PP2A), have major regulatory roles to play and may be potential targets for the design of new therapeutic agents.

Asthma↗

PubMed2006

Proteomic analysis reveals a novel role for the actin cytoskeleton in vincristine resistant childhood leukemia--an in vivo study.

Intrinsic or acquired resistance to vincristine (VCR), an antimicrotubule agent used in the treatment of childhood acute lymphoblastic leukemia (ALL), is a major clinical problem. Using a clinically relevant NOD/SCID mouse xenograft model of ALL, we established that alterations in the actin and tubulin cytoskeleton are involved in in vivo VCR resistance. Altered protein expression between VCR-sensitive ALL xenografts, and xenografts with intrinsic or acquired VCR resistance, was identified using 2-D DIGE coupled with MS. Of the 19 proteins displaying altered expression, 11 are associated with the actin cytoskeleton. Altered expression of the actin- and/or tubulin-binding proteins gelsolin, moesin, ezrin, tropomyosin, CAP-G, HSP27, HSP70, TCP-1, and stathmin were associated with in vivo VCR resistance. The actin-regulating protein gelsolin was increased in both acquired and resistant leukemia as confirmed by immunoblotting and gene expression. The major cytoskeletal protein, gamma-actin, was down-regulated in the VCR-resistant leukemia xenografts; in contrast, there was no significant change in beta-actin expression. This study provides the first evidence for a role of the actin cytoskeleton in intrinsic and acquired in vivo antimicrotubule drug resistance in childhood leukemia and highlights the power of 2-D DIGE for the discovery of resistance markers, pharmacoproteomics, and signaling pathways in cancer.

Actins↗

PubMed2006

Clinical proteomics: present and future prospects.

Advances in proteomics technology offer great promise in the understanding and treatment of the molecular basis of disease. The past decade of proteomics research, the study of dynamic protein expression, post-translational modifications, cellular and sub-cellular protein distribution, and protein-protein interactions, has culminated in the identification of many disease-related biomarkers and potential new drug targets. While proteomics remains the tool of choice for discovery research, new innovations in proteomic technology now offer the potential for proteomic profiling to become standard practice in the clinical laboratory. Indeed, protein profiles can serve as powerful diagnostic markers, and can predict treatment outcome in many diseases, in particular cancer. A number of technical obstacles remain before routine proteomic analysis can be achieved in the clinic; however the standardisation of methodologies and dissemination of proteomic data into publicly available databases is starting to overcome these hurdles. At present the most promising application for proteomics is in the screening of specific subsets of protein biomarkers for certain diseases, rather than large scale full protein profiling. Armed with these technologies the impending era of individualised patient-tailored therapy is imminent. This review summarises the advances in proteomics that has propelled us to this exciting age of clinical proteomics, and highlights the future work that is required for this to become a reality.

Journal Article↗

PubMed2005

Tissue-specific tropomyosin isoform composition.

Four distinct genes encode tropomyosin (Tm) proteins, integral components of the actin microfilament system. In non-muscle cells, over 40 Tm isoforms are derived using alternative splicing. Distinct populations of actin filaments characterized by the composition of these Tm isoforms are found differentially sorted within cells (Gunning et al. 1998b). We hypothesized that these distinct intracellular compartments defined by the association of Tm isoforms may allow for independent regulation of microfilament function. Consequently, to understand the molecular mechanisms that give rise to these different microfilaments and their regulation, a cohort of fully characterized isoform-specific Tm antibodies was required. The characterization protocol initially involved testing the specificity of the antibodies on bacterially produced Tm proteins. We then confirmed that these Tm antibodies can be used to probe the expression and subcellular localization of different Tm isoforms by Western blot analysis, immunofluorescence staining of cells in culture, and immunohistochemistry of paraffin wax-embedded mouse tissues. These Tm antibodies, therefore, have the capacity to monitor specific actin filament populations in a range of experimental systems.

Actins↗

PubMed2004

Neuronal-associated microtubule proteins class III beta-tubulin and MAP2c in neuroblastoma: role in resistance to microtubule-targeted drugs.

Advanced stage neuroblastoma has a poor clinical outcome and microtubule-destabilizing agents, such as the Vinca alkaloids, are an important component in the treatment of this childhood cancer. Vinca alkaloids bind to beta-tubulin on the alpha/beta-tubulin heterodimer and disrupt microtubule dynamics, leading to cell death. To date, studies examining the contribution of microtubules and associated proteins to the efficacy of microtubule-destabilizing agents in neuroblastoma have been limited. In this study, BE2-C neuroblastoma cells previously selected for resistance to either vincristine (BE/VCR10) or colchicine (BE/CHCb0.2) were found to display significant decreases in neuronal-specific class III beta-tubulin. Interestingly, vincristine-selected cells exhibited increased levels of polymerized tubulin that were not due to alpha-tubulin and class I, II, or III beta-tubulin mutations. Expression levels of the microtubule-depolymerizing protein stathmin were significantly increased in BE/VCR10 cells. In contrast, levels of MAP2a and MAP2b were relatively unaltered. A marked decrease in the neuronal protein, MAP2c, was identified in the vincristine-selected cells and, to a lesser extent, in the colchicine-selected cells. This is the first report describing specific microtubule alterations in neuroblastoma cells resistant to tubulin-targeted agents. The results indicate a need to identify the factors responsible for resistance to tubulin-targeted agents in neuroblastoma so that improved and novel treatment strategies can be developed for this drug refractory disease.

Drug Resistance, Neoplasm↗

PubMed2003

Proteome analysis of vinca alkaloid response and resistance in acute lymphoblastic leukemia reveals novel cytoskeletal alterations.

Vinca alkaloids are used widely in the treatment of both childhood and adult cancers. Their cellular target is the beta-tubulin subunit of alpha/beta-tubulin heterodimers, and they act to inhibit cell division by disrupting microtubule dynamics. Despite the effectiveness of these agents, drug resistance is a major clinical problem. To identify the underlying mechanisms behind vinca alkaloid resistance, we have performed high resolution differential proteome analysis. Treatment of drug-sensitive human leukemia cells (CCRF-CEM) with vincristine identified numerous proteins involved in the cellular response to vincristine. In addition, differential protein expression was analyzed in leukemia cell lines selected for resistance to vincristine (CEM/VCR R) and vinblastine (CEM/VLB100). This combined proteomic approach identified 10 proteins altered in both vinca alkaloid response and resistance: beta-tubulin, alpha-tubulin, actin, heat shock protein 90beta, 14-3-3tau, 14-3-3epsilon, L-plastin, lamin B1, heterogeneous nuclear ribonuclear protein-F, and heterogeneous nuclear ribonuclear protein-K. Several of these proteins have not previously been associated with drug resistance and are thus novel targets for elucidation of resistance mechanisms. In addition, seven of these proteins are associated with the tubulin and/or actin cytoskeletons. This study provides novel insights into the interrelationship between the microtubule and microfilament systems in vinca alkaloid resistance.

Actins↗

PubMed2003

A fluorescent natural product for ultra sensitive detection of proteins in one-dimensional and two-dimensional gel electrophoresis.

Lightning Fast is a sensitive fluorescence-based stain for detecting proteins in one-dimensional and two-dimensional polyacrylamide electrophoresis gels. It contains the fluorophore epicocconone from the fungus Epicoccum nigrum that interacts noncovalently with sodium dodecyl sulfate and protein. Stained proteins can be excited optimally by near-ultraviolet light of about 395 nm or with visible light of about 520 nm. The stain can be excited using a range of sources used in image analysis systems including UVA (ca. 365 nm) and UVB (ca. 302 nm) transilluminators; Xenon-arc lamps; 488 nm and 457 nm Argon-ion lasers; 473 nm and 532 nm neodymium: yttrium aluminum garnet (Nd:YAG) solid-state lasers; 543 nm helium-neon lasers, and emerging violet, blue and green diode lasers. Maximum fluorescence emission of the dye is at approximately 610 nm. The limit of detection in one-dimensional gels stained with Lightning Fast protein gel stain is less than 100 pg of protein, rivaling the current limits of matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). Lightning Fast was found to be considerably more sensitive than SYPRO Ruby, SYPRO Orange, silver and Coomassie Brilliant Blue G-250 in matched experiments. Staining takes as little as 3.5 h and stained proteins displayed quantitative linearity over more than four orders of magnitude, thereby allowing visualization of entire proteomes. Lightning Fast protein gel staining is compatible with subsequent peptide mass fingerprinting using MALDI-MS and Edman-based sequencing chemistry.

Animals↗

PubMed2003

Microtubule alterations and mutations induced by desoxyepothilone B: implications for drug-target interactions.

Epothilones, like paclitaxel, bind to beta-tubulin and stabilize microtubules. We selected a series of four leukemia sublines that display increasing levels of resistance to the epothilone analog desoxyepothilone B (dEpoB). The dEpoB cells selected in 30-140 nM were approximately 15-fold cross-resistant to paclitaxel, while 300 nM selected cells were 467-fold resistant to this agent. The dEpoB-selected cells are hypersensitive to microtubule destabilizing agents, and express increased levels of class III beta-tubulin and MAP4. A novel class I beta-tubulin mutation, A231T, that affects microtubule stability but does not alter paclitaxel binding, was identified. The 300 nM selected cells acquired a second mutation, Q292E, situated near the M loop of class I beta-tubulin. These cells fail to undergo drug-induced tubulin polymerization due to dramatically reduced drug binding. The dEpoB-resistant leukemia cells provide novel insights into microtubule dynamics and, in particular, drug-target interactions.

Antineoplastic Agents↗

PubMed2003

Drug resistance mechanisms in cancer cells: a proteomics perspective.

Chemotherapy forms a significant component of the treatment of many human cancers. The failure of tumor cells to respond to the action of cytotoxic drugs is referred to as drug resistance. Mechanisms mediating resistance are multifaceted and often not clearly defined. The challenge of elucidating the link between drug response and resistance, and then integrating the information for the purpose of therapeutic intervention, is a major task that awaits scientists in the post-genomic era. Rapid advances in proteome technologies have made it possible to simultaneously identify multiple proteins involved in drug refractory cancers. This methodology has the potential to isolate novel proteins, including those associated with resistance to specific drugs and those involved in conferring broad resistance to a range of compounds. The identification of key proteins will lead to the possibility of developing molecular, biological or pharmaceutical strategies to modify the action of such proteins when their inappropriate structure or expression is contributing to drug-resistant disease.

Antineoplastic Agents↗