[Show abstract][Hide abstract] ABSTRACT: Cancer cells can be drug resistant due to genetic variation at multiple steps in the drug response pathway, including drug efflux pumping, target mutation, and blunted apoptotic response. These are not discriminated by conventional cell survival assays. Here, we report a rapid and convenient high-content cell-imaging assay that measures multiple physiological changes in cells responding to antimitotic small-molecule drugs. Our one-step, no-wash assay uses three dyes to stain living cells and is much more accurate for scoring weakly adherent mitotic and apoptotic cells than conventional antibody-based assays. We profiled responses of 33 cell lines to 8 antimitotic drugs at multiple concentrations and time points using this assay and deposited our data and assay protocols into a public database (http://lincs.hms.harvard.edu/). Our data discriminated between alternative mechanisms that compromise drug sensitivity to paclitaxel and revealed an unexpected bell-shaped dose-response curve for BI2536, a highly selective inhibitor of Polo-like kinases. Our approach can be generalized, is scalable, and should therefore facilitate identification of molecular biomarkers for mechanisms of drug insensitivity in high-throughput screens and other assays.
Preview · Article · Jun 2013 · Journal of Biomolecular Screening
[Show abstract][Hide abstract] ABSTRACT: Small molecule inhibitors of Kinesin-5 (K5Is) that arrest cells in mitosis with monopolar spindles are promising anti-cancer drug candidates. Clinical trials of K5Is revealed dose-limiting neutropenia, or loss of neutrophils, for which the molecular mechanism is unclear. We investigated the effects of a K5I on HL60 cells, a human promyelocytic leukemia cell line that is often used to model dividing neutrophil progenitors in cell culture. We found K5I treatment caused unusually rapid death of HL60 cells exclusively during mitotic arrest. This mitotic death occurred via the intrinsic apoptosis pathway with molecular events that include cytochrome c leakage into the cytoplasm, caspase activation, and Parp1 cleavage. Bcl-2 overexpression protected from death. We probed mitochondrial physiology to find candidate triggers of cytochrome c release, and observed a decrease of membrane potential (ΔΨm) before mitochondrial outer membrane permeabilization (MOMP). Interestingly, this loss of ΔΨm was not blocked by overexpressing Bcl-2, suggesting it might be a cause of Bax/Bak activation, not a consequence. Taken together, these results show that K5I induces intrinsic apoptosis during mitotic arrest in HL60 with loss of ΔΨm as an upstream event of MOMP.
[Show abstract][Hide abstract] ABSTRACT: Kinesin-5 inhibitors (K5I) are promising antimitotic cancer drug candidates. They cause prolonged mitotic arrest and death of cancer cells, but their full range of phenotypic effects in different cell types has been unclear. Using time-lapse microscopy of cancer and normal cell lines, we find that a novel K5I causes several different cancer and noncancer cell types to undergo prolonged arrest in monopolar mitosis. Subsequent events, however, differed greatly between cell types. Normal diploid cells mostly slipped from mitosis and arrested in tetraploid G(1), with little cell death. Several cancer cell lines died either during mitotic arrest or following slippage. Contrary to prevailing views, mitotic slippage was not required for death, and the duration of mitotic arrest correlated poorly with the probability of death in most cell lines. We also assayed drug reversibility and long-term responses after transient drug exposure in MCF7 breast cancer cells. Although many cells divided after drug washout during mitosis, this treatment resulted in lower survival compared with washout after spontaneous slippage likely due to chromosome segregation errors in the cells that divided. Our analysis shows that K5Is cause cancer-selective cell killing, provides important kinetic information for understanding clinical responses, and elucidates mechanisms of drug sensitivity versus resistance at the level of phenotype.
Full-text · Article · Dec 2008 · Molecular Cancer Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Although this talk will not be about thermodynamics, it is useful to recall the spirit of the classical Carnot analysis of heat engines. The Carnot analysis tells us that, for specified constraints (upper and lower operating temperatures), there is a limit to how much of a supplied entity (heat) can be converted into a desired product (work). Although highly idealized Carnot engines cannot be realized in practice, they nevertheless play an essential role in the analysis, not least in establishing that the derived bound on conversion of heat to work is sharp: To the extent that Carnot engines might be approximated in the limit by real engines, the theoretical Carnot bound on conversion can be realized arbitrarily closely. This is of enormous importance, for the Carnot analysis offers a theoretical benchmark against which all engines, subject to the same operating constraints, can be measured.
Process designs (like engine designs) are finally judged on practical grounds, but the Carnot story is an instructive one, suggesting a spirit in which reactor-separator synthesis might be approached. Can we know when a candidate steady-state design is, in some theoretical sense, efficient in its production rate relative to all other designs consistent with the same commitment of resources? In particular, is there a theoretical limit to what might have been produced from the same feed in any steady-state design that utilizes the same reactor size as the candidate design, that respects the same pressure-temperature bounds within reactor units, and that respects the same constraints on the production of unwanted side products? More generally, for specified temperature-pressure bounds in reactor units is there a minimum reactor size, independent of design, that is required to achieve a target productivity, and, if so, how might it be calculated? And are there certain universal Carnot-like configurations that invariably achieve maximum productivity subject to process constraints?
Some thoughts on these questions will be presented.
[Show abstract][Hide abstract] ABSTRACT: The classical Carnot analysis provides means to calculate, relative to a specified heat supply and relative to specified temperature bounds, a limit on the work that can be obtained in any cyclic process consistent with those specifications. Moreover, the Carnot analysis indicates that the calculated limit is sharp, to the extent that it can be attained by a special process (a Carnot cycle) which cannot itself be realized but which can be approximated arbitrarily closely by actual processes. We indicate how, in the spirit of the Carnot analysis, theoretical limits to the productivity of steady-state reactor−separator systems can be calculated relative to specified commitments of resources and relative to specified constraints. We also indicate a sense in which those limits are sharp, to the extent that they might be attained by certain idealized reactor−separator systems which, like Carnot cycles, cannot be realized in practice but which might be approached by actual processes.
No preview · Article · Jul 2007 · Industrial & Engineering Chemistry Research
[Show abstract][Hide abstract] ABSTRACT: Much attention has been paid recently to bistability and switch-like behavior that might be resident in important biochemical reaction networks. There is, in fact, a great deal of subtlety in the relationship between the structure of a reaction network and its capacity to engender bistability. In common physicochemical settings, large classes of extremely complex networks, taken with mass action kinetics, cannot give rise to bistability no matter what values the rate constants take. On the other hand, bistable behavior can be induced in those same settings by certain very simple and classical mass action mechanisms for enzyme catalysis of a single overall reaction. We present a theorem that distinguishes between those mass action networks that might support bistable behavior and those that cannot. Moreover, we indicate how switch-like behavior results from a well-studied mechanism for the action of human dihydrofolate reductase, an important anti-cancer target.
Full-text · Article · Jul 2006 · Proceedings of the National Academy of Sciences