The development of resistance to targeted therapeutics is a challenging issue for the treatment of cancer. Cancers that have mutations in BRCA, a DNA repair protein, have been treated with poly (ADP-ribose) polymerase (PARP) inhibitors, which target a second DNA repair mechanism with the aim of inducing synthetic lethality. While these inhibitors have shown promise clinically, the development of resistance can limit their effectiveness as a therapy. This study investigated mechanisms of resistance in BRCA-mutated cancer cells (HCC1937) to Olaparib (AZD2281) using TRACER, a technique for measuring dynamics of transcription factor (TF) activity in living cells. TF activity was monitored in the parental HCC1937 cell line and two distinct resistant cell lines, one with restored wild-type BRCA1 and one with acquired resistance independent of BRCA1 for 48 hours during treatment with Olaparib. Partial least squares discriminant analysis (PLSDA) was used to categorize the three cell types based on TF activity, and network analysis was used to investigate the mechanism of early response to Olaparib in the study cells. NOTCH signaling was identified as a common pathway linked to resistance in both Olaparib-resistant cell types. Western blotting confirmed upregulation of NOTCH protein, and sensitivity to Olaparib was restored through co-treatment with a gamma secretase inhibitor. The identification of NOTCH signaling as a common pathway contributing to PARP inhibitor resistance by TRACER indicates the efficacy of transcription factor dynamics in identifying targets for intervention in treatment-resistant cancer and provides a new method for determining effective strategies for directed chemotherapy.
Compared with conventional coagulation tests and factor-specific assays, viscoelastic hemostatic assays (VHAs) can provide a more thorough evaluation of clot formation and lysis but have several limitations including clot deformation. In this proof-of-concept study, we test a noncontact technique, termed resonant acoustic rheometry (RAR), for measuring the kinetics of human plasma coagulation. Specifically, RAR utilizes a dual-mode ultrasound technique to induce and detect surface oscillation of blood samples without direct physical contact and measures the resonant frequency of the surface oscillation over time, which is reflective of the viscoelasticity of the sample. Analysis of RAR results of normal plasma allowed defining a set of parameters for quantifying coagulation. RAR detected a flat-line tracing of resonant frequency in hemophilia A plasma that was corrected with the addition of tissue factor. Our RAR results captured the kinetics of plasma coagulation and the newly defined RAR parameters correlated with increasing tissue factor concentration in both healthy and hemophilia A plasma. These findings demonstrate the feasibility of RAR as a novel approach for VHA, providing the foundation for future studies to compare RAR parameters to conventional coagulation tests, factor-specific assays, and VHA parameters.
To examine the possible physiological significance of Mrp, a multi-subunit cation/proton antiporter from Vibrio cholerae, a chromosomal deletion Δmrp of V. cholerae was constructed and characterized. The resulting mutant showed a consistent early growth defect in LB broth that became more evident at elevated pH of the growth medium and increasing Na+ or K+ loads. After 24 h incubation, these differences disappeared likely due to the concerted effort of other cation pumps in the mrp mutant. Phenotype MicroArray analyses revealed an unexpected systematic defect in nitrogen utilization in the Δmrp mutant that was complemented by using the mrpA'-F operon on an arabinose-inducible expression vector. Deletion of the mrp operon also led to hypermotility, observable on LB and M9 semi-solid agar. Surprisingly, Δmrp mutation resulted in wild-type biofilm formation in M9 despite a growth defect but the reverse was true in LB. Furthermore, the Δmrp strain exhibited higher susceptibility to amphiphilic anions. These pleiotropic phenotypes of the Δmrp mutant demonstrate how the chemiosmotic activity of Mrp contributes to the survival potential of V. cholerae despite the presence of an extended battery of cation/proton antiporters of varying ion selectivity and pH profile operating in the same membrane.
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