Expression and function of drug transporters in primitive CML cells

Chronic myeloid leukaemia (CML) is a stem cell (SC) disorder initiated by the reciprocal translocation between chromosome 9 and 22, giving rise to the Philadelphia (Ph) chromosome and the resulting expression of the oncogenic fusion protein BCR-ABL. The current first line of treatment is imatinib mesylate (IM), a tyrosine kinase inhibitor (TKI) that competes with ATP to block ABL kinase activity, which in turn prevents tyrosine phosphorylation of downstream molecules and selectively induces apoptosis of BCR-ABL cells. However, despite excellent cytogenetic responses, only a minority of patients achieve complete molecular response (CMR). We have previously identified a population of quiescent (q) Ph+ SC found in chronic phase (CP) CML that are relatively insensitive to IM and other TKIs and which may be responsible for the molecular persistence of this disease. This population may be insensitive because TKIs do not reach therapeutic concentrations within the cell. Such resistance to classical chemotherapeutic drugs, the phenomenon of multidrug resistance (MDR), is mediated by ABC transporters. In this study we have investigated whether CML SC express the clinically relevant ABC transporters and determine their interaction with TKIs. In addition, we determined whether the inhibition of these transporters increased the efficacy of TKI against CML SC. Using CML CD34+ cells isolated from newly diagnosed patients, normal CD34+ cells and cell lines transduced with specific transporters as controls, the relative expression of drug transporters were determined in CML CD34+ cells and intracellular staining confirmed protein expression. The interaction of drug transporters with TKIs was assessed using a combination of substrate displacement assays and radiolabelled assays. The effect of transporter inhibitors 3 with TKIs on the growth and differentiation of q34+ and more mature CD34+ cells from CML patients in CP were assessed with regard to cell division, apoptosis and BCR-ABL kinase activity. When compared to normal CD34+ cells, CML CD34+ cells over-expressed ABCG2 mRNA. In contrast MDR1 expression was reduced in CML CD34+ cells and MRP1 was detected at similar expression levels in both populations. All three drug transporters were expressed at the protein levels in CML CD34+ cells. It was determined that at therapeutic concentrations (5μM) IM and nilotinib both inhibited ABCG2 and MDR1 and nilotinib also inhibited MRP1. Neither drug was a substrate for any of the transporters. In contrast, dasatinib was shown to be a substrate for ABCG2 and MRP1, but had no effect on MDR1. Therefore activity and concentration of dasatinib but not IM or nilotinib may be altered by the activity of these proteins. In keeping with their inhibitory activity, neither IM nor nilotinib demonstrated significantly increased efficacy when combined with specific ABC transporter inhibitors (FTC or PSC 833). Surprisingly, although dasatinib was a substrate for ABCG2 and MRP1, dasatinib did not further increase apoptosis, or reduce the qSC population. Therefore, although MDR1, MRP1 and ABCG2 were found to be expressed and functional in CML CD34+ cells and to interact with TKI, the co-treatment of TKIs with drug transporter inhibitors did not further increase apoptosis, reduce BCRABL kinase activity or reduce the qSC population. Therefore, modulation of individual transporter activity is unlikely to reverse the resistance of this population of cells to TKI and will not improve the clinical response to these drugs

Inhibition of MDR1 does not sensitize primitive chronic myeloid leukemia CD34⁺ cells to imatinib

<p><b>Objective:</b> To investigate the interaction of imatinib mesylate (IM) with the clinically relevant adenosine triphosphate-binding cassette efflux transporter MDR1 (ABCB1) in cells from patients with chronic myeloid leukemia (CML) and to explore whether inhibition of this transporter would improve IM's efficacy in the elimination of CML CD34<sup>+</sup> cells by increasing cell-associated drug accumulation.</p> <p><b>Materials and Methods:</b> Cells from newly diagnosed chronic-phase CML patients were harvested by leukapheresis and enriched to >95% CD34<sup>+</sup>. Expression of the transporter gene MDR1 was performed by quantitative reverse transcription polymerase chain reaction. Interaction of IM with MDR1 was analyzed by substrate (rhodamine 123) displacement assay. Cell-associated levels of IM in CML CD34<sup>+</sup> cells were measured by high-pressure liquid chromatography. Intracellular phospho-CrkL levels, apoptosis in total CML CD34<sup>+</sup> cells and high-resolution tracking of cell division were assayed by flow cytometry.</p> <p><b>Results:</b> Measurements of cell-associated IM uptake showed significantly lower drug levels in CD34<sup>+</sup> cells, particularly the CD38<sup>-</sup> subpopulation, as compared to IM-sensitive K562 cells. MDR1 was expressed at low level and dye efflux studies demonstrated very little MDR1 activity in CML CD34<sup>+</sup> cells. Furthermore, combination treatment of primitive CML cells with IM and the MDR1 inhibitor PSC833 did not result in elevated cell-associated IM levels. Although we observed slightly enhanced cytostasis with IM when combined with PSC833, this was independent of BCR-ABL inhibition because no associated decrease in phospho-CrkL was observed.</p> <p><b>Conclusions:</b> Our findings demonstrate that inhibition of MDR1 neither enhances the effect of IM against BCR-ABL activity, nor significantly potentiates IM's efficiency in eliminating primitive CML cells.</p&gt

Polymorphic Elements of the Human IL-6 Gene in Gastric Cancer

There has been much interest focusing on the contribution of cytokines to the genetic variation known to occur between the immune responses of different individuals. Interleukin-6 (IL-6), originally defined as a B cell growth factor, was rapidly identified as under-pinning the induction of the acute-phase response and being heavily involved in inflammatory responses generally. More recently, the role of IL-6 in human malignancy has become a topic of interest. The aim of this study was to examine four polymorphic elements, three in the promoter 5'region and one VNTR in the 3'region and define the relationships of these alleles in a West of Scotland control population. The relationships were then observed in two malignant cohorts; patients diagnosed with Gastric carcinoma and patients diagnosed with Barrett's Oesophagus, to determine possible disease related associations. Investigation of an African American cohort and subsequent rheumatoid individuals also provided a comparison in a different ethnic group and the associations of the polymorphic elements in an autoimmune disease. Digestion of amplified DNA determined the polymorphisms in the 5' promoter region and PAGE separation resolved the minisatellite alleles at the 3' VNTR. The data revealed that strong significant associations between the alleles form three prominent 5' promoter haplotype families and four dominant extended haplotypes, which include the 3'VNTR in the West-of-Scotland population. In the gastric carcinoma cohort the 4 polymorphisms were similarly distributed to the control population and did not prove to be genetic markers relating to this disease. Similarly no statistical significance was detected with the Barrett's Oesophagus cohort. However, the presence of H.pylori in the malignant cohorts proved to be of significant importance in the distribution the 5' haplotype families. The haplotype IL6.01 was significantly more prominent in H.Pylori positive patients of the gastric cohort, where as the haplotype IL6.02 was more prominent in the Barrett's Oesophagus patients. In the African American population, no statistical difference was detected between the control cohort and the Rheumatoid arthritis patients. The results reported here demonstrate that markers cannot necessarily be considered independent of one another across human cytokine genes. Additionally, where markers fall in, or close to, functional elements such as transcription factor binding sites, it should be remembered that other polymorphic elements might be contributing to the observed effect

Solvation Dynamics in Protein Environments Studied by Photon Echo Spectroscopy

Photon echo spectroscopy is used to study the mechanisms of solvation dynamics in protein environments at room temperature. Ultrafast and additional multi-exponential long time scales are observed in the three-pulse photon echo peak shift data of the fluorescein dye eosin bound to lysozyme in aqueous solution. The dynamics of the solvated lysozyme are characterized with dielectric continuum models that integrate dielectric data for water with that for lysozyme. By comparing our data with previous results for eosin in water [Lang, M. J.; Jordanides, X. J.; Song, X.; Fleming, G. R. J. Chem. Phys. 1999, 110, 5584], we find that the total coupling of the electronic transition frequency of eosin to the nuclear motions of the aqueous lysozyme solution is smaller than in the aqueous solution. On an ultrafast time scale, solvation appears to be dominated by the surrounding water and not by the ultrafast internal motions of lysozyme. However, over long time scales, lysozyme does contribute significantly, either directly through motions of polar side chains or indirectly through reorientation of the water “bound” to the surface of the protein

The mechanism of energy transfer in the bacterial photosynthetic reaction center

In the accompanying paper (Scholes, G. D.; Jordanides, X. J.; Fleming, G. R. J. Phys. Chem. 2001, 105, 1640, a generalization of Förster theory is developed to calculate electronic energy transfer (EET) in molecular aggregates. Here we apply the theory to wild-type and mutant photosynthetic reaction centers (RCs) from Rb. sphaeroides, as well as to the wild-type RC from Rps. Viridis. Experimental information from the X-ray crystallographic structure, resonance Raman excitation profiles, and hole-burning measurements are integrated with calculated electronic couplings to model the EET dynamics within the RC complex. Optical absorption and circular dichroism spectra are calculated at various temperatures between 10 K and room temperature, and compare well with the experimentally observed spectra. The calculated rise time of the population of the lower exciton state of P, P -, as a result of energy transfer from the accessory bacteriochlorophyll, B, to the special pair, P, in Rb. sphaeroides (Rps. Viridis) wild-type at 298 K is 193 fs (239 fs), and is in satisfactory agreement with experimental results. Our calculations, which employ a weak-coupling mechanism suggest that the upper exciton state of P, P + plays a central role in trapping excitation from B. Our ability to predict the experimental rates is partly attributed to a proper calculation of the spectral overlap J δR ( ) using the vibronic progressions. The main advance we have made, however, is to calculate the electronic couplings V δR in terms of the molecular composition of donor and/or acceptor aggregates, rather than treating the acceptors P + and P -as point dipoles associated with each spectroscopic band. Thus, we believe our electronic couplings capture the essence of the many-body interactions within the RC. Calculations for EET in two mutants, (M)-L214H (the beta mutant) and (M)H202L (the heterodimer), are in reasonable agreement with experimental results. In the case of the heterodimer the agreement depends on a decrease in the electronic couplings between D M and the rest of the pigments

Uptake of synthetic low density lipoprotein by leukemic stem cells — a potential stem cell targeted drug delivery strategy

Chronic Myeloid Leukemia (CML) stem/progenitor cells, which over-express Bcr-Abl, respond to imatinib by a reversible block in proliferation without significant apoptosis. As a result, patients are unlikely to be cured owing to the persistence of leukemic quiescent stem cells (QSC) capable of initiating relapse. Previously, we have reported that intracellular levels of imatinib in primary primitive CML cells (CD34<sup>+</sup>38<sup>lo/−</sup>), are significantly lower than in CML progenitor cells (total CD34<sup>+</sup>) and leukemic cell lines. The aim of this study was to determine if potentially sub-therapeutic intracellular drug concentrations in persistent leukemic QSC may be overcome by targeted drug delivery using synthetic Low Density Lipoprotein (sLDL) particles. As a first step towards this goal, however, the extent of uptake of sLDL by leukemic cell lines and CML patient stem/progenitor cells was investigated. Results with non-drug loaded particles have shown an increased and preferential uptake of sLDL by Bcr-Abl positive cell lines in comparison to Bcr-Abl negative. Furthermore, CML CD34<sup>+</sup> and primitive CD34<sup>+</sup>38<sup>lo/−</sup> cells accumulated significantly higher levels of sLDL when compared with non-CML CD34<sup>+</sup> cells. Thus, drug-loading the sLDL nanoparticles could potentially enhance intracellular drug concentrations in primitive CML cells and thus aid their eradication

Hydrogen Bond Dynamics Near A Micellar Surface: Origin of the Universal Slow Relaxation at Complex Aqueous Interfaces

The dynamics of hydrogen bonds among water molecules themselves and with the polar head groups (PHG) at a micellar surface have been investigated by long molecular dynamics simulations. The lifetime of the hydrogen bond between a PHG and a water molecule is found to be much longer than that between any two water molecules, and is likely to be a general feature of hydrophilic surfaces of organized assemblies. Analyses of individual water trajectories suggest that water molecules can remain bound to the micellar surface for more than a hundred picosecond. The activation energy for such a transition from the bound to a free state for the water molecules is estimated to be about 3.5kcal/mole.Comment: 12 pages. Phys. Rev. Lett. (Accepted) (2002

Chronic myeloid leukemia stem cells are not dependent on Bcr-Abl kinase activity for their survival

Recent evidence suggests CML stem cells are insensitive to kinase inhibitors and responsible for minimal residual disease in treated patients. We investigated whether CML stem cells, in a transgenic mouse model of CML-like disease or derived from patients, are dependent on Bcr-Abl. In the transgenic model, following re-transplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off, were able to persist in vivo and re-initiate leukemia in secondary recipients upon Bcr-Abl re-expression. Bcr-Abl knockdown in human CD34+ CML cells cultured for 12 days in physiological growth factors achieved partial inhibition of Bcr-Abl and downstream targets p-CrkL and p-STAT5, inhibition of proliferation and colony forming cells, but no reduction of input cells. The addition of dasatinib further inhibited p-CrkL and p-STAT5, yet only reduced input cells by 50%. Complete growth factor withdrawal plus dasatinib further reduced input cells to 10%, however the surviving fraction was enriched for primitive leukemic cells capable of growth in long-term culture initiating cell assay and expansion upon removal of dasatinib and addition of growth factors. Together these data suggest that CML stem cell survival is Bcr-Abl kinase independent and suggest curative approaches in CML must focus on kinase-independent mechanisms of resistance