Biomedical subjects

Maria Kavallaris

Publications and source records attributed to Maria Kavallaris.

14 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

Acid-cleavable polymeric core-shell particles for delivery of hydrophobic drugs.

Here we describe the combined use of acid-labile microgel approach and RAFT-mediated seeded dispersion polymerization technique to prepare an acid-cleavable core-shell like polymeric colloidal system for the delivery of hydrophobic drugs at slightly acidic sites. A new bisacrylate acetal crosslinker was copolymerized with n-butyl acrylate (BA) in the presence of a RAFT agent using a dispersion polymerization technique, which yielded crosslinked spherical particles with the size ranging between 150 and 500 nm. The particles were cleaved in a pH-dependent manner similar to the acid-labile hydrolysis behaviour of the crosslinker. In order to mask the hydrophobic surface of the particles, polyethylene glycol acrylate (PEG-A) was grafted onto poly(BA) seed particles via the RAFT agent groups on the particle surface. The acidic-site selective delivery potential of the poly(BA)-g-poly(PEG-A) particles was assessed in-vitro using a lipophilic fluorescent dye as a model hydrophobic drug. Ca. 73% and 34% of the total dye loaded in the particles was found to be released at pH 5.0 and 7.4 in 24 h, respectively. The growth of human neuroblastoma cells was not affected by the incubation with the core-shell particles and their cleavage by-products up to 3 mg/ml concentration. The physicochemical and the functional features support the potential value of the acid-cleavable poly(BA) core-poly(PEG-A) shell particles as carriers for the delivery of hydrophobic drugs at acidic sites.

Acetates↗

PubMed2006

The estrogen-responsive B box protein is a novel regulator of the retinoid signal.

Retinoic acid (RA) induces growth arrest, cell death, and differentiation in many human cancer cells in vitro and has entered routine clinical use for the treatment of several human cancer types. One mechanism by which cancer cells evade retinoid-induced effects is through repression of retinoic acid receptor beta (RARbeta) gene transcription. The RA response element beta (betaRARE) is the essential DNA sequence required for retinoid-induced RARbeta transcription. Here we show that the estrogen-responsive B box protein (EBBP), a member of the RING-B box-coiled-coil protein family, is a betaRARE-binding protein. EBBP undergoes serine threonine phosphorylation and enhanced protein stability after RA treatment. Following RA treatment, we also observed increased nuclear EBBP levels in aggregates with the promyelocytic leukemia protein at promyelocytic leukemia nuclear bodies. EBBP enhanced RA-responsive RARbeta transcription in RA-sensitive and -resistant cancer cells, which were resistant to both a histone deacetylase inhibitor and a demethylating agent. EBBP-specific small interfering RNA reduced basal and RA-induced RARbeta expression. EBBP increased betaRARE-transactivating function through its coiled-coil domain. Taken together, our work suggests that EBBP may have a pivotal role in the retinoid anti-cancer signal.

Amino Acid Sequence↗

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

A novel plant toxin, persin, with in vivo activity in the mammary gland, induces Bim-dependent apoptosis in human breast cancer cells.

Phytochemicals have provided an abundant and effective source of therapeutics for the treatment of cancer. Here we describe the characterization of a novel plant toxin, persin, with in vivo activity in the mammary gland and a p53-, estrogen receptor-, and Bcl-2-independent mode of action. Persin was previously identified from avocado leaves as the toxic principle responsible for mammary gland-specific necrosis and apoptosis in lactating livestock. Here we used a lactating mouse model to confirm that persin has a similar cytotoxicity for the lactating mammary epithelium. Further in vitro studies in a panel of human breast cancer cell lines show that persin selectively induces a G2-M cell cycle arrest and caspase-dependent apoptosis in sensitive cells. The latter is dependent on expression of the BH3-only protein Bim. Bim is a sensor of cytoskeletal integrity, and there is evidence that persin acts as a microtubule-stabilizing agent. Due to the unique structure of the compound, persin could represent a novel class of microtubule-targeting agent with potential specificity for breast cancers.

Animals↗

PubMed2005

Proteomics and disease: opportunities and challenges.

Since the human genome was sequenced, there has been intense activity to understand the function of the 30,000 identified genes; attention has now turned to the products of genes--proteins. Proteomics is the large-scale study of the structure and function of proteins; it includes the rapidly evolving field of disease proteomics, which aims to identify proteins involved in human disease and to understand how their expression, structure and function cause illness. Proteomics has identified proteins that offer promise as diagnostic or prognostic markers, or as therapeutic targets in a range of illnesses, including cancer, immune rejection after transplantation, and infectious diseases such as tuberculosis and malaria; it has the potential to allow patient-tailored therapy. Some major challenges remain, both technical (eg, detecting "low-abundance" proteins, and maintaining sample stability) and in data management (eg, correlating changes in proteins with disease processes).

Databases, Factual↗

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

Effects of MYCN antisense oligonucleotide administration on tumorigenesis in a murine model of neuroblastoma.

BACKGROUND: Human MYCN (hMYCN) oncogene amplification is a powerful predictor of treatment failure in childhood neuroblastoma, and dysregulation of hMYCN protein expression appears to be critically involved in the pathogenesis of this disease. We used hMYCN antisense (AS) oligonucleotides to investigate, both in vitro and in vivo, the therapeutic potential of inhibiting hMYCN expression. METHODS: We transiently transfected human neuroblastoma IMR-32 cells, which have an amplified hMYCN gene, with fluorescently labeled hMYCN AS or scrambled (SCR) control oligonucleotides and used fluorescence-activated cell sorting to enrich for cell populations containing different levels of the oligonucleotides. We used fluorescence immunocytochemistry or reverse transcription polymerase chain reaction to assay gene expression levels and trypan blue exclusion to assay growth inhibition in the cell populations. We examined the effects of continuous treatment for 6 weeks with AS or SCR oligonucleotides via subcutaneously implanted microosmotic pumps on tumor growth in a transgenic mouse model of hMYCN-induced neuroblastoma (n = 20 mice per group). All statistical tests were two-sided. RESULTS: IMR-32 cells treated with AS oligonucleotides had approximately half as much hMYCN protein and cell proliferation as either SCR oligonucleotide-transfected or mock-transfected controls; the differences were statistically significant. Transgenic mice treated with AS oligonucleotides had lower tumor incidence and statistically significantly lower tumor mass than SCR-treated or untreated control mice. Compared with control treatments, AS oligonucleotide treatment in vitro and in vivo was associated with decreased expression of hMYCN and putative hMYCN target genes but not with that of closely related genes. Several AS oligonucleotide-treated mice developed tumors contralateral to the site of oligonucleotide administration, whereas SCR oligonucleotide-treated or untreated mice displayed bilateral tumor growth. CONCLUSIONS: Decreased expression of hMYCN protein is achievable with the use of AS oligonucleotide treatment, even in the presence of hMYCN oncogene amplification. Antisense strategies targeting the hMYCN oncogene in vivo decrease mouse neuroblastoma tumorigenesis. Investigation of their clinical effect in children with neuroblastoma is warranted.

Animals↗

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

Growth inhibitory retinoid effects after recruitment of retinoid X receptor beta to the retinoic acid receptor beta promoter.

Nuclear retinoid receptors mediate retinoid effects through tissue-specific, ligand-receptor interactions and subsequent transcriptional regulation of secondary target genes. Retinoic acid receptor beta (RARbeta) is itself a retinoid target gene with a retinoic acid response element (betaRARE) in the 5' untranslated region of the RARbeta2 gene. Altered transcriptional regulation of RARbeta may play a role in human carcinogenesis and the retinoid-responsiveness of malignant cells. Here we used retinoid X receptor-specific antibodies in electrophoretic mobility shift assays to show that the retinoid X receptor beta (RXRbeta) protein was recruited to the betaRARE, after retinoid treatment of retinoid-sensitive neuroblastoma (NB), lung and breast cancer cell lines, but not retinoid-resistant lung and breast cancer cell lines. RXRbeta selectively enhanced retinoid-induced transcriptional activation of the betaRARE. Stable overexpression of RXRalpha and RXRbeta in NB cells resulted in marked growth inhibition and cell death, which increased after retinoid treatment. However, only proteins from the RXRbeta transfectants exhibited specific RXRbeta binding to the betaRARE in vitro and in vivo, enhanced histone acetylation and increased endogenous RARbeta expression. These data indicate that recruitment of RXRbeta to the betaRARE, and consequent induction of endogenous RARbeta expression, is an important component in the retinoid anticancer signal. RXRalpha may also participate in the retinoid signal, but through mechanisms that do not involve RARbeta.

Acetylation↗

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↗

PubMed2002

Microtubule alterations and resistance to tubulin-binding agents (review).

Antimitotic agents that bind to tubulin and disrupt microtubule dynamics, such as vinca alkaloids and taxanes, are an integral part of the treatment of many human cancers. Unfortunately the clinical success of these agents can be severely hindered by the emergence of drug resistant tumour cells. In recent years interest has focused on alterations in the cellular target of antimicrotubule compounds, the tubulin/ microtubule system, and their role in drug resistance. This review will discuss tubulin/microtubule alterations associated with resistance to tubulin-binding agents. Novel antimicrotubule compounds that hold promise in the treatment of drug-resistant disease and are currently undergoing clinical evaluation will also be reviewed.

Animals↗