P-glycoprotein and breast cancer resistance protein in acute myeloid leukaemia cells treated with the Aurora-B Kinase Inhibitor barasertib-hQPA

<p>Abstract</p> <p>Background</p> <p>Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies. Over-expression of the ABC drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in acute myeloid leukaemia (AML). Barasertib-hQPA is a highly selective inhibitor of aurora-B kinase that has shown tumouricidal activity against a range tumour cell lines including those of leukaemic AML origin.</p> <p>Methods</p> <p>Effect of barasertib-hQPA on the pHH3 biomarker and cell viability was measured in a panel of leukaemic cell lines and 37 primary AML samples by flow cytometry. Pgp status was determined by flow cytometry and BCRP status by flow cytometry and real-time PCR.</p> <p>Results</p> <p>In this study we report the creation of the cell line OCI-AML3DNR, which over-expresses Pgp but not BCRP or multidrug resistance-associated protein (MRP), through prolonged treatment of OCI-AML3 cells with daunorubicin. We demonstrate that Pgp (OCI-AML3DNR and KG-1a) and BCRP (OCI-AML6.2) expressing AML cell lines are less sensitive to barasertib-hQPA induced pHH3 inhibition and subsequent loss of viability compared to transporter negative cell lines. We also show that barasertib-hQPA resistance in these cell lines can be reversed using known Pgp and BCRP inhibitors. We report that barasertib-hQPA is not an inhibitor of Pgp or BCRP, but by using ¹⁴[C]-barasertib-hQPA that it is effluxed by these transporters. Using phosphoHistone H3 (pHH3) as a biomarker of barasertib-hQPA responsiveness in primary AML blasts we determined that Pgp and BCRP positive primary samples were less sensitive to barasertib-hQPA induced pHH3 inhibition (p = <0.001) than samples without these transporters. However, we demonstrate that IC₅₀inhibition of pHH3 by barasertib-hQPA was achieved in 94.6% of these samples after 1 hour drug treatment, in contrast to the resistance of the cell lines.</p> <p>Conclusion</p> <p>We conclude that Pgp and BCRP status and pHH3 down-regulation in patients treated with barasertib should be monitored in order to establish whether transporter-mediated efflux is sufficient to adversely impact on the efficacy of the agent.</p

Kinetochore-Independent Chromosome Poleward Movement during Anaphase of Meiosis II in Mouse Eggs

Kinetochores are considered to be the key structures that physically connect spindle microtubules to the chromosomes and play an important role in chromosome segregation during mitosis. Due to different mechanisms of spindle assembly between centrosome-containing mitotic cells and acentrosomal meiotic oocytes, it is unclear how a meiotic spindle generates the poleward forces to drive two rounds of meiotic chromosome segregation to achieve genome haploidization. We took advantage of the fact that DNA beads are able to induce bipolar spindle formation without kinetochores and studied the behavior of DNA beads in the induced spindle in mouse eggs during meiosis II. Interestingly, DNA beads underwent poleward movements that were similar in timing and speed to the meiotic chromosomes, although all the beads moved together to the same spindle pole. Disruption of dynein function abolished the poleward movements of DNA beads but not of the meiotic chromosomes, suggesting the existence of different dynein-dependent and dynein-independent force generation mechanisms for the chromosome poleward movement, and the latter may be dependent on the presence of kinetochores. Consistent with the observed DNA bead poleward movement, sperm haploid chromatin (which also induced bipolar spindle formation after injection to a metaphase egg without forming detectable kinetochore structures) also underwent similar poleward movement at anaphase as DNA beads. The results suggest that in the chromatin-induced meiotic spindles, kinetochore attachments to spindle microtubules are not absolutely required for chromatin poleward movements at anaphase

The functional asymmetry of auditory cortex is reflected in the organization of local cortical circuits

The primary auditory cortex (A1) is organized tonotopically, with neurons sensitive to high and low frequencies arranged in a rostro-caudal gradient. We used laser scanning photostimulation in acute slices to study the organization of local excitatory connections onto layers 2 and 3 (L2/3) of the mouse A1. Consistent with the organization of other cortical regions, synaptic inputs along the isofrequency axis (orthogonal to the tonotopic axis) arose predominantly within a column. By contrast, we found that local connections along the tonotopic axis differed from those along the isofrequency axis: some input pathways to L3 (but not L2) arose predominantly out-of-column. In vivo cell-attached recordings revealed differences between the sound-responsiveness of neurons in L2 and L3. Our results are consistent with the hypothesis that auditory cortical microcircuitry is specialized to the one-dimensional representation of frequency in the auditory cortex