Contrasting effects of diclofenac and ibuprofen on active imatinib uptake into leukaemic cells.

BACKGROUND: The human organic cation transporter-1 (OCT-1) is the primary active protein for imatinib uptake into target BCR-ABL-positive cells. Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used by chronic myeloid leukaemia (CML) patients on imatinib to manage musculoskeletal complaints. METHODS: Here we investigated the impact of NSAIDs on functional activity of the OCT-1 (OCT-1 activity; OA) in CML cells. RESULTS: Although ten of twelve NSAIDs tested had no significant impact on OA (P>0.05), we observed increased OA (27% increase in K562; 22% increase in KU812 cells, P<0.05) and reduced IC50(imatinib) when treated with diclofenac. Co-incubation with imatinib and diclofenac resulted in a significantly lower viable cell number compared with imatinib alone. In contrast, ibuprofen led to a significant decrease in OA, an increase in IC50(imatinib) and thus reduced the cytotoxicity of imatinib. In primary CML samples, diclofenac significantly increased OA, particularly in patients with low OA (<4 ng per 200 000 cells), and significantly decreased IC50(imatinib). Ibuprofen induced significant decreases in OA in CML samples and healthy donors. CONCLUSION: On the basis of the expected impact of these two drugs on OA, ibuprofen should be avoided in combination with imatinib. Further studies are warranted regarding the potential benefit of diclofenac to improve OA in a clinical setting.

Rule-based modelling of conjugative plasmid transfer and incompatibility.

COSMIC-rules, an individual-based model for bacterial adaptation and evolution, has been used to study virtual transmission of plasmids within bacterial populations, in an environment varying between supportive and inhibitory. The simulations demonstrate spread of antibiotic resistance (R) plasmids, both compatible and incompatible, by the bacterial gene transfer process of conjugation. This paper describes the behaviour of virtual plasmids, their modes of exchange within bacterial populations and the impact of antibiotics, together with the rules governing plasmid transfer. Three case studies are examined: transfer of an R plasmid within an antibiotic-susceptible population, transfer of two incompatible R plasmids and transfer of two compatible R plasmids. R plasmid transfer confers antibiotic resistance on recipients. For incompatible plasmids, one or other plasmid could be maintained in bacterial cells and only that portion of the population acquiring the appropriate plasmid-encoded resistance survives exposure to the antibiotics. By contrast, the compatible plasmids transfer and mix freely within the bacterial population that survives in its entirety in the presence of the antibiotics. These studies are intended to inform models for examining adaptive evolution in bacteria. They provide proof of principle in simple systems as a platform for predicting the behaviour of bacterial populations in more complex situations, for example in response to changing environments or in multi-species bacterial assemblages.

Rule-based computing system for microbial interactions and communications: evolution in virtual bacterial populations.

We have developed a novel rule-based computing system of microbial interactions and communications, referred to as COSMIC-Rules, for simulating evolutionary processes within populations of virtual bacteria. The model incorporates three levels: the bacterial genome, the bacterial cell and an environment inhabited by such cells. The virtual environment in COSMIC-Rules can contain multiple substances, whose relative toxicity or nutrient status is specified by the genome of the bacterium. Each substance may be distributed uniformly or in a user-defined manner. The organisms in COSMIC-Rules possess individually-defined physical locations, size, cell division status and genomes. Genes and/or gene systems are represented by abstractions that may summate sometimes complex phenotypes. Central to COSMIC-Rules is a simplified representation of bacterial species, each containing a functional genome including, where desired, extrachromosomal elements such as plasmids and/or bacteriophages. A widely applicable computer representation of biological recognition systems based on bit string matching is essential to the model. This representation permits, for example, the modelling of protein-protein interactions, receptor-ligand interactions and DNA-DNA transactions. COSMIC-Rules is intended to inform studies on bacterial adaptation and evolution, and to predict behaviour of populations of pathogenic bacteria and their viruses. The framework is constructed for parallel execution across a large number of machines and efficiently utilises a 64 processor development cluster. It will run on any Grid system and has successfully tested simulations with millions of bacteria, of multiple species and utilising multiple substrates. The model may be used for large-scale simulations where a genealogical record for individual organisms is required.

The clinical significance of ABCB1 overexpression in predicting outcome of CML patients undergoing first-line imatinib treatment.

Tyrosine kinase inhibitor (TKI) therapy results in excellent responses in the majority of patients with chronic myeloid leukaemia. First-line imatinib treatment, with selective switching to nilotinib when patients fail to meet specific molecular targets or for imatinib intolerance, results in excellent overall molecular responses and survival. However, this strategy is less effective in cases of primary imatinib resistance; moreover, 25% of patients develop secondary resistance such that 20-35% of patients initially treated with imatinib will eventually experience treatment failure. Early identification of these patients is of high clinical relevance. Since the drug efflux transporter ABCB1 has previously been implicated in TKI resistance, we determined if early increases in ABCB1 mRNA expression (fold change from diagnosis to day 22 of imatinib therapy) predict for patient response. Indeed, patients exhibiting a high fold rise (⩾2.2, n=79) were significantly less likely to achieve early molecular response (BCR-ABL1IS ⩽10% at 3 months; P=0.001), major molecular response (P<0.0001) and MR4.5 (P<0.0001). Additionally, patients demonstrated increased levels of ABCB1 mRNA before the development of mutations and/or progression to blast crisis. Patients with high fold rise in ABCB1 mRNA were also less likely to achieve major molecular response when switched to nilotinib therapy (49% vs 82% of patients with low fold rise). We conclude that early evaluation of the fold change in ABCB1 mRNA expression may identify patients likely to be resistant to first- and second-generation TKIs and who may be candidates for alternative therapy.

The Paton individual-based model legacy.

Ray Paton oversaw the creation of a long lineage of Individual-based Models (IbMs) and this paper discusses the five most successful. All of these concern the development of adaptation, covering both evolutionary time and organism lifetime (somatic time). Of the five models discussed here, the first is based on a plant-herbivore model, the other four are based on a substrate-bacteria model, with the option of antibiotics.

Detection of two further beta-type cytochromes in Rhodopseudomonas spheroides.

The photosynthetically-incompetent mutant V-2 of Rhodopseudomonas spheroides which is incapable of synthesising bacteriochlorophyll was grown aerobically under conditions of both high and low aeration. Potentiometric titration a at 560 nm minus 570 nm revealed the presence of several different components tentatively identified as b-type cytochromes. Two such components of oxidation-reduction midpoint potentials of +390 mV +/- 10 mV and +255 mV +/- 7mV have not previously been detected in membranes of Rps. spheroides. These components have also been resolved by difference spectra at controlled oxidation-reduction potentials and fourth derivative spectra. Neither component appeared to react with CO. With increasing aeration of the culture medium the relative concentration of these two b-type cytochromes diminished, whilst that of the a-type oxidase increased.

Rule-based simulation of temperate bacteriophage infection: restriction-modification as a limiter to infection in bacterial populations.

An individual-based model (IbM) for bacterial adaptation and evolution, COSMIC-Rules, has been employed to simulate interactions of virtual temperate bacteriophages (phages) and their bacterial hosts. Outcomes of infection mimic those of a phage such as lambda, which can enter either the lytic or lysogenic cycle, depending on the nutritional status of the host. Infection of different hosts possessing differing restriction and modification systems is also simulated. Phages restricted upon infection of one restricting host can be adapted (by host-controlled modification of the phage genome) and subsequently propagate with full efficiency on this host. However, such ability is lost if the progeny phages are passaged through a new host with a different restriction and modification system before attempted re-infection of the original restrictive host. The simulations show that adaptation and re-adaptation to a particular host-controlled restriction and modification system result in lower efficiency and delayed lysis of bacterial cells compared with infection of non-restricting host bacteria. Such biologically realistic simulations validate the use of the IbM approach to predicting behaviour of bacteriophages in bacterial populations. The applicability of the model for more complex scenarios aimed at predictive modelling of bacterial evolution in a changing environment and the implications for the spread of viruses in a wider context are discussed.

Chronic myeloid leukemia CD34+ cells have reduced uptake of imatinib due to low OCT-1 activity.

Active influx of imatinib in chronic myeloid leukemia (CML) cells is mediated by the organic cation transporter 1 (OCT-1). Functional activity of OCT-1 (OCT-1 Activity) in mononuclear cells is an excellent predictor of molecular response over the first 24 months of imatinib therapy for chronic phase patients. CML progenitor cells are less sensitive to imatinib-induced apoptosis and are likely contributors to disease persistence. We investigated whether alterations in the expression and function of OCT-1 have a role in imatinib resistance in progenitors. We found the intracellular uptake and retention (IUR) of imatinib, OCT-1 Activity and OCT-1 mRNA expression are all significantly lower in CML CD34+ cells compared with mature CD34- cells (P<0.001). However, no differences in IUR or OCT-1 Activity were observed between these subsets in healthy donors. In contrast to OCT-1, ABCB1 and ABCG2 seemed to have no functional role in the transport of imatinib in CML CD34+ cells. Consistent with the observation that nilotinib uptake is not OCT-1 dependent, the IUR of nilotinib did not differ between CML CD34+ and CD34- cells. These results indicate that low imatinib accumulation in primitive CML cells, mediated through reduced OCT-1 Activity may be a critical determinant of long-term disease persistence.

Blocking cytokine signaling along with intense Bcr-Abl kinase inhibition induces apoptosis in primary CML progenitors.

In chronic myeloid leukemia (CML) cell lines, brief exposure to pharmacologically relevant dasatinib concentrations results in apoptosis. In this study, we assess the impact of intensity and duration of Bcr-Abl kinase inhibition on primary CD34(+) progenitors of chronic phase CML patients. As CML cells exposed to dasatinib in vivo are in a cytokine-rich environment, we also assessed the effect of cytokines (six growth factors cocktail or granulocyte-macrophage colony-stimulating factor (CSF) or granulocyte-CSF) in combination with dasatinib. In the presence of cytokines, short-term intense Bcr-Abl kinase inhibition (>or=90% p-Crkl inhibition) with 100 nM dasatinib did not reduce CD34(+) colony-forming cells (CFCs). In contrast, without cytokines, short-term exposure to dasatinib reduced CML-CD34(+) CFCs by 70-80%. When cytokines were added immediately after short-term exposure to dasatinib, CML-CD34(+) cells remained viable, suggesting that oncogene dependence of these cells can be overcome by concomitant or subsequent exposure to cytokines. Additional inhibition of Janus tyrosine kinase (Jak) activity re-established the sensitivity of CML progenitors to intense Bcr-Abl kinase inhibition despite the presence of cytokines. These findings support the contention that therapeutic strategies combining intense Bcr-Abl kinase inhibition and blockade of cytokine signaling pathways can be effective for eradication of CML progenitors.

Uptake of gentamicin by Staphylococcus aureus possessing gentamicin-modifying enzymes: enhancement of uptake by puromycin and N,N'-dicyclohexylcarbodiimide.

Uptake of gentamicin by a gentamicin-resistant strain of Staphylococcus aureus possessing the aminoglycoside-modifying phosphotransferase enzyme APH(2") was enhanced by the protein synthesis inhibitor, puromycin or by the proton-translocating ATPase inhibitor, N,N'-dicylohexylcarbodiimide. Such enhanced uptake was inhibited by carbonyl cyanide p trifluoromethoxyphenylhydrazone or by valinomycin in the presence of potassium ions, suggesting a role for the transmembrane proton motive force in the process. The accumulated gentamicin did not cause loss of cell viability and exhibited altered chromatographic mobility compared with a control (unmodified) preparation of gentamicin.

Accumulation of gentamicin by Staphylococcus aureus: the role of the transmembrane electrical potential.

Accumulation of gentamicin by gentamicin-susceptible Staphylococcus aureus was examined by using the ionophorous antibiotic, valinomycin and the protontranslocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide. The effects of these inhibitors on the transmembrane electrical potential (delta psi) were determined by measuring the equilibrium distribution of the tetraphenylphosphonium ion. The results indicate a direct correlation between delta psi and the extent of gentamicin uptake. However, under conditions where a significant delta psi existed across the plasma membrane, uptake of gentamicin was negligible. A threshold delta psi may thus be required to initiate gentamicin uptake. The proposed threshold delta psi appears to vary depending upon the external concentration of gentamicin.

The pericentromeric heterochromatin of the grass Zingeria biebersteiniana (2n = 4) is composed of Zbcen1-type tandem repeats that are intermingled with accumulated dispersedly organized sequences.

DNA reassociation and hydroxyapatite chromatography were used to isolate high-copy DNA of the grass Zingeria biebersteiniana (2n = 4). In situ hybridization demonstrated that the DNA isolated was enriched for pericentromere-specific repetitive sequences. One abundant pericentromere-specific component is the differentially methylated tandem-repeat family Zbcen1. Other sequences isolated, Zb46 and Zb47A, are dispersed and display similarity to parts of the gypsy- and copia-like retrotransposable elements of other grasses. In situ hybridization with the copia-like sequence Zb47A resulted in dispersed labelling along the chromosome arms, with a significant signal accumulation in the pericentromeric region of all chromosomes. It is concluded that the pericentromeric heterochromatin of Z. biebersteiniana is composed of members of the Zbcen1 tandem repeat family and that these tandem arrays are intermingled with accumulated putative copia-like retrotransposon sequences. An observed Rab1 interphase orientation suggests that the length of the chromosomes rather than the genome size is the determining factor of the Rab1 phenomenon.