Reduced expression of p27 is a novel mechanism of docetaxel resistance in breast cancer cells

INTRODUCTION: Docetaxel is one of the most effective chemotherapeutic agents in the treatment of breast cancer. Breast cancers can have an inherent or acquired resistance to docetaxel but the causes of this resistance remain unclear. However, apoptosis and cell cycle regulation are key mechanisms by which most chemotherapeutic agents exert their cytotoxic effects. METHODS: We created two docetaxel-resistant human breast cancer cell lines (MCF-7 and MDA-MB-231) and performed cDNA microarray analysis to identify candidate genes associated with docetaxel resistance. Gene expression changes were validated at the RNA and protein levels by reverse transcription PCR and western analysis, respectively. RESULTS: Gene expression cDNA microarray analysis demonstrated reduced p27 expression in docetaxel-resistant breast cancer cells. Although p27 mRNA expression was found to be reduced only in MCF-7 docetaxel-resistant sublines (2.47-fold), reduced expression of p27 protein was noted in both MCF-7 and MDA-MB-231 docetaxel-resistant breast cancer cells (2.83-fold and 3.80-fold, respectively). CONCLUSIONS: This study demonstrates that reduced expression of p27 is associated with acquired resistance to docetaxel in breast cancer cells. An understanding of the genes that are involved in resistance to chemotherapy may allow further development in modulating drug resistance, and may permit selection of those patients who are most likely to benefit from such therapies

A Novel Tool for Studying Auxin-Metabolism: The Inhibition of Grapevine Indole-3-Acetic Acid-Amido Synthetases by a Reaction Intermediate Analogue

An important process for the regulation of auxin levels in plants is the inactivation of indole-3-acetic acid (IAA) by conjugation to amino acids. The conjugation reaction is catalysed by IAA-amido synthetases belonging to the family of GH3 proteins. Genetic approaches to study the biological significance of these enzymes have been hampered by large gene numbers and a high degree of functional redundancy. To overcome these difficulties a chemical approach based on the reaction mechanism of GH3 proteins was employed to design a small molecule inhibitor of IAA-amido synthetase activity. Adenosine-5′-[2-(1H-indol-3-yl)ethyl]phosphate (AIEP) mimics the adenylated intermediate of the IAA-conjugation reaction and was therefore proposed to compete with the binding of MgATP and IAA in the initial stages of catalysis. Two grapevine IAA-amido synthetases with different catalytic properties were chosen to test the inhibitory effects of AIEP in vitro. GH3-1 has previously been implicated in the grape berry ripening process and is restricted to two amino acid substrates, whereas GH3-6 conjugated IAA to 13 amino acids. AIEP is the most potent inhibitor of GH3 enzymes so far described and was shown to be competitive against MgATP and IAA binding to both enzymes with Ki-values 17-68-fold lower than the respective Km-values. AIEP also exhibited in vivo activity in an ex planta test system using young grape berries. Exposure to 5–20 µM of the inhibitor led to decreased levels of the common conjugate IAA-Asp and reduced the accumulation of the corresponding Asp-conjugate upon treatment with a synthetic auxin. AIEP therefore represents a novel chemical probe with which to study IAA-amido synthetase function

Reporter Proteins in Whole-Cell Optical Bioreporter Detection Systems, Biosensor Integrations, and Biosensing Applications

Whole-cell, genetically modified bioreporters are designed to emit detectable signals in response to a target analyte or related group of analytes. When integrated with a transducer capable of measuring those signals, a biosensor results that acts as a self-contained analytical system useful in basic and applied environmental, medical, pharmacological, and agricultural sciences. Historically, these devices have focused on signaling proteins such as green fluorescent protein, aequorin, firefly luciferase, and/or bacterial luciferase. The biochemistry and genetic development of these sensor systems as well as the advantages, challenges, and common applications of each one will be discussed

Functional Characterization of Circulating Tumor Cells with a Prostate-Cancer-Specific Microfluidic Device

Cancer metastasis accounts for the majority of cancer-related deaths owing to poor response to anticancer therapies. Molecular understanding of metastasis-associated drug resistance remains elusive due to the scarcity of available tumor tissue. Isolation of circulating tumor cells (CTCs) from the peripheral blood of patients has emerged as a valid alternative source of tumor tissue that can be subjected to molecular characterization. However, issues with low purity and sensitivity have impeded adoption to clinical practice. Here we report a novel method to capture and molecularly characterize CTCs isolated from castrate-resistant prostate cancer patients (CRPC) receiving taxane chemotherapy. We have developed a geometrically enhanced differential immunocapture (GEDI) microfluidic device that combines an anti-prostate specific membrane antigen (PSMA) antibody with a 3D geometry that captures CTCs while minimizing nonspecific leukocyte adhesion. Enumeration of GEDI-captured CTCs (defined as intact, nucleated PSMA+/CD45− cells) revealed a median of 54 cells per ml identified in CRPC patients versus 3 in healthy donors. Direct comparison with the commercially available CellSearch® revealed a 2–400 fold higher sensitivity achieved with the GEDI device. Confocal microscopy of patient-derived GEDI-captured CTCs identified the TMPRSS2:ERG fusion protein, while sequencing identified specific androgen receptor point mutation (T868A) in blood samples spiked with only 50 PC C4-2 cells. On-chip treatment of patient-derived CTCs with docetaxel and paclitaxel allowed monitoring of drug-target engagement by means of microtubule bundling. CTCs isolated from docetaxel-resistant CRPC patients did not show any evidence of drug activity. These measurements constitute the first functional assays of drug-target engagement in living circulating tumor cells and therefore have the potential to enable longitudinal monitoring of target response and inform the development of new anticancer agents

Epithelial-mesenchymal transition and cancer stem cells: a dangerously dynamic duo in breast cancer progression

Aberrant activation of a latent embryonic program - known as the epithelial-mesenchymal transition (EMT) - can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence. The induction of EMT entails the loss of epithelial characteristics and the de novo acquisition of a mesenchymal phenotype. In breast cancer, the EMT state has been associated with cancer stem cell properties including expression of the stem cell-associated CD44+/CD24-/low antigenic profile, self-renewal capabilities and resistance to conventional therapies. Intriguingly, EMT features are also associated with stem cells isolated from the normal mouse mammary gland and human breast reduction tissues as well as the highly aggressive metaplastic and claudin-low breast tumor subtypes. This has implications for the origin of these breast tumors as it remains unclear whether they derive from cells that have undergone EMT or whether they represent an expansion of a pre-existing stem cell population that expresses EMT-associated markers to begin with. In the present review, we consider the current evidence connecting EMT and stem cell attributes and discuss the ramifications of these newly recognized links for our understanding of the emergence of distinct breast cancer subtypes and breast cancer progression

Biotin protein ligase from Saccharomyces cerevisiae / by Steven William Polyak.

Bibliography: leaves 130-159.159, [61] leaves, [7] leaves of plates : ill. (some col.) ; 30 cm.Investigates the physical and catalytic properties of a biotin protein ligase and aims to understand further the interaction of this enzyme with its protein substrates.Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 2000

Biotin Protein Ligase Is a Target for New Antibacterials

There is a desperate need for novel antibiotic classes to combat the rise of drug resistant pathogenic bacteria, such as Staphylococcus aureus. Inhibitors of the essential metabolic enzyme biotin protein ligase (BPL) represent a promising drug target for new antibacterials. Structural and biochemical studies on the BPL from S. aureus have paved the way for the design and development of new antibacterial chemotherapeutics. BPL employs an ordered ligand binding mechanism for the synthesis of the reaction intermediate biotinyl-5′-AMP from substrates biotin and ATP. Here we review the structure and catalytic mechanism of the target enzyme, along with an overview of chemical analogues of biotin and biotinyl-5′-AMP as BPL inhibitors reported to date. Of particular promise are studies to replace the labile phosphoroanhydride linker present in biotinyl-5′-AMP with alternative bioisosteres. A novel in situ click approach using a mutant of S. aureus BPL as a template for the synthesis of triazole-based inhibitors is also presented. These approaches can be widely applied to BPLs from other bacteria, as well as other closely related metabolic enzymes and antibacterial drug targets

Hepatitis C infection and alcohol use: A dangerous mix for the liver and antiviral immunity

This article presents the proceedings of a symposium presented at the meeting of the Research Society on Alcoholism, held in Santa Barbara, California, in June 2005. The organizers and chairs were Sam Zakhari and Gyongyi Szabo. The presentations included (1) Mitochondrial Abnormalities Induced by Hepatitis C -Alcohol Interaction by Steven Weinman; (2) Effects of Acute and Chronic Ethanol on Innate Antiviral Signaling Pathways, Hepatitis C Replication, and Human Liver Cell Transcription by Stephen Polyak; (3) Ethanol Alters Dendritic Cell Function In Vivo and Impairs the Subsequent Cellular Immune Responses to Hepatitis C Proteins by Costica Aloman; and (4) Pathogenic Interactions Between Hepatitis C Virus and Alcohol Use in Humans: Dendritic Cells as Common Targets by Gyongyi Szabo. This symposium summarizes the state of knowledge of cellular and molecular pathways by which alcohol and HCV have pathogenic interactions resulting in depression of the immune response and liver damage in chronic HCV infection