In clinical trials, the tyrosine kinase inhibitor STI571 has proven highly effective in reducing leukemic cell burden in chronic myeloid leukemia (CML). The overall sensitivity of CML CD34 ؉ progenitor cells to STI571 and the degree to which cell death was dependent on cell cycle status were determined. Stem cells (Lin ؊ CD34 ؉ ) from the peripheral blood of patients with CML in chronic phase and from granulocyte-colony-stimulating factor-mobilized healthy donors were labeled with carboxy-fluorescein diacetate succinimidyl diester dye to enable highresolution tracking of cell division. Then they were cultured for 3 days with and without growth factors ؎ STI571. After culture, the cells were separated by fluorescence-activated cell sorting into populations of viable quiescent versus cycling cells for genotyping. For healthy controls, in the presence of growth factors, STI571 affected neither cell cycle kinetics nor recovery of viable cells. In the absence of growth factors, normal cells were unable to divide. For CML samples, in the presence or absence of growth factors, the response to STI571 was variable. In the most sensitive cases, STI571 killed almost all dividing cells; however, a significant population of viable CD34 ؉ cells was recovered in the undivided peak and confirmed to be part of the leukemic clone. STI571 also appeared to exhibit antiproliferative activity on the quiescent population. These studies confirm that CML stem cells remain viable in a quiescent state even in the presence of growth factors and STI571. Despite dramatic short-term responses in vivo, such in vitro insensitivity to STI571, in combination with its demonstrated antiproliferative activity, could translate into disease relapse after prolonged therapy. IntroductionChronic myeloid leukemia (CML) is a clonal myeloproliferative disease characterized by the t(9;22) chromosome translocation that, in turn, creates the BCR-ABL oncogene. [1][2][3] The fusion gene product is a p210 oncoprotein containing a constitutively active tyrosine kinase that confers certain growth advantages to the Philadelphia-positive (Ph ϩ ) clone compared with normal hematopoietic cells. 4 We have demonstrated recently the existence of a population of rare, primitive, quiescent stem cells in all chronic-phase CML patient samples, whether derived from peripheral blood or bone marrow. These stem cells are predominantly Ph ϩ , express high levels of CD34 ϩ but lack the markers CD38, CD45RA, or CD71, and can spontaneously exit G 0 to enter a continuously proliferating state, either in vitro or to produce Ph ϩ progeny in immunocompromised mice in vivo. 5,6 Many cancers are treated with relatively nonselective cytotoxic drugs that affect normal and malignant cells. Because most available chemotherapeutic agents show some degree of S-phase specificity, cells that are not actively dividing may prove resistant to such drugs. This raises the possibility that the quiescent leukemic cells we have identified in patients with CML are likely to survive standard chemotherapy r...

Dasatinib (BMS-354825), a novel dual SRC/BCR-ABL kinase inhibitor, exhibits greater potency than imatinib mesylate (IM) and inhibits the majority of kinase mutations in IM-resistant chronic myeloid leukemia (CML). We have previously demonstrated that IM reversibly blocks proliferation but does not induce apoptosis of primitive CML cells. Here, we have attempted to overcome this resistance with dasatinib. Primitive IM-resistant CML cells showed only single-copy BCR-ABL but expressed significantly higher BCR-ABL transcript levels and BCR-ABL protein compared with more mature CML cells (P ‫؍‬ .031). In addition, CrKL phosphorylation was higher in the primitive CD34 ؉ CD38 ؊ than in the total CD34 ؉ population (P ‫؍‬ .002). In total CD34 ؉ CML cells, IM inhibited phosphorylation of CrKL at 16 but not 72 hours, consistent with enrichment of an IM-resistant primitive population. CD34 ؉ CD38 ؊ CML cells proved resistant to IM-induced inhibition of CrKL phosphorylation and apoptosis, whereas dasatinib led to significant inhibition of CrKL phosphorylation. Kinase domain mutations were not detectable in either IM or dasatinib-resistant primitive CML cells. These data confirm that dasatinib is more effective than IM within the CML stem cell compartment; however, the most primitive quiescent CML cells appear to be inherently resistant to both drugs. ( IntroductionChronic myeloid leukemia (CML) is a clonal hemopoietic disorder that is sustained by a population of primitive and transplantable stem cells. 1 These stem cells are Philadelphia chromosome positive (Ph ϩ ) and express the oncogenic tyrosine kinase BCR-ABL. 2,3 BCR-ABL is central to the pathogenesis of CML, and mutation of critical elements leads to a reduction in transformation potential. 4,5 In the malignant clone, BCR-ABL is constitutively active, resulting in autophosphorylation of the kinase domain and of downstream substrates including CrKL. 6,7 The specificity of CrKL phosphorylation to BCR-ABL signaling, partnered with stability of the phosphoprotein complex, has led to its acceptance as an excellent method to assess BCR-ABL status. [8][9][10] Imatinib mesylate (IM) has been introduced as first-line targeted therapy for CML. IM is a tyrosine kinase inhibitor that is relatively specific for BCR-ABL. 11 In vitro, in Ph ϩ cell lines and in bulk cultures of primary CML cells, IM reverses the autophosphorylation of BCR-ABL and inhibits phosphorylation of downstream targets including CrKL. 11,12 Within 48 to 72 hours of IM exposure, CML cells undergo apoptosis. More recently, Chu et al 13 have confirmed that CrKL phosphorylation is also inhibited by IM in CD34-enriched populations of primary CML cells. However, our own work and that of others confirms that primitive CML cells do not readily undergo apoptosis, even after prolonged in vitro exposure to the drug. [14][15][16][17] The link between inactivation of BCR-ABL kinase activity and induction of apoptosis in the most primitive CML cells therefore remains unclear.In vivo, even in chronic phase, clinical respon...

Recent evidence suggests chronic myeloid leukemia (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, after retransplantation, donor-derived CML stem cells in which Bcr-Abl expression had been induced and subsequently shut off were able to persist in vivo and reinitiate leukemia in secondary recipients on Bcr-Abl reexpression. Bcr-Abl knockdown in human CD34(+) CML cells cultured for 12 days in physiologic 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 a long-term culture-initiating cell assay and expansion on 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.

Chronic myeloid leukemia (CML) stem and progenitor cells overexpress BcrAbl and are insensitive to imatinib mesylate (IM). We therefore investigated whether these cells were efficiently targeted by nilotinib. In K562, the inhibitory concentration (IC50) of nilotinib was 30 nM versus 600 nM for IM, consistent with its reported 20-fold-higher potency. However, in primary CD34(+) CML cells, nilotinib and IM were equipotent for inhibition of BcrAbl activity, producing equivalent but incomplete reduction in CrkL phosphorylation at 5 microM. CML CD34(+) cells were still able to expand over 72 hours with 5 microM of either drug, although there was a concentration-dependent restriction of amplification. As for IM, the most primitive cells (CFSE(max)) persisted and accumulated over 72 hours with nilotinib and remained caspase-3 negative. Furthermore, nilotinib with IM led to further accumulation of this population, suggesting at least additive antiproliferative effects. These results confirmed that, like IM, the predominant effect of nilotinib is antiproliferative rather than proapoptotic.

Superhydrophobic surfaces are present in nature on the leaves of many plant species. Water rolls on these surfaces, and the rolling motion picks up particles including bacteria and viruses. Man-made superhydrophobic surfaces have been made in an effort to reduce biofouling. We show here that the anti-biofouling property of a superhydrophobic surface is due to an entrapped air-bubble layer that reduces contact between the bacteria and the surface. Further, we showed that prolonged immersion of superhydrophobic surfaces in water led to loss of the bubble-layer and subsequent bacterial adhesion that unexpectedly exceeded that of the control materials. This behavior was not restricted to one particular type of material but was evident on different types of superhydrophobic surfaces. This work is important in that it suggests that superhydrophobic surfaces may actually encourage bacterial adhesion during longer term exposure.

Clostridium difficile is a major nosocomial pathogen and the main causative agent of antibiotic-associated diarrhoea. The organism produces two potent toxins, A and B, which are its major virulence factors. These are chromosomally encoded on a region termed the pathogenicity locus (PaLoc), which also contains regulatory genes, and is absent in non-toxigenic strains. Here we show that the PaLoc can be transferred from the toxin-producing strain, 630Δerm, to three non-toxigenic strains of different ribotypes. One of the transconjugants is shown by cytotoxicity assay to produce toxin B at a similar level to the donor strain, demonstrating that a toxigenic C. difficile strain is capable of converting a non-toxigenic strain to a toxin producer by horizontal gene transfer. This has implications for the treatment of C. difficile infections, as non-toxigenic strains are being tested as treatments in clinical trials.

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The prevalence of healthcare-associated infection caused by multidrugresistant bacteria is of critical concern worldwide. It is reported on the development of a bactericidal surface prepared by use of a simple, upscalable, two-step dipping strategy to incorporate crystal violet and di(octyl)-phosphinic-acid-capped zinc oxide nanoparticles into medical grade silicone, as a strategy to reduce the risk of infection. The material is characterized by UV-vis absorbance spectroscopy, X-ray photoelectron spectroscopy (XPS), inductively coupled plasma-optical emission spectroscopy (ICP-OES) and transmission electron microscopy (TEM) and confi rmed the incorporation of the ZnO nanoparticles in the polymer. The novel system proves to be a highly versatile bactericidal material when tested against both Staphylococcus aureus and Escherichia coli , key causative micro-organisms for hospitalacquired infection (HAI). Potent antimicrobial activity is noted under dark conditions, with a signifi cant enhancement exhibits when the surfaces are illuminated with a standard hospital light source. This polymer has the potential to decrease the risk of HAI, by killing bacteria in contact with the surface.