Generation of Superoxide by purified and relipidated cytochrome b559 in the absence of cytosolic activators

AbstractPurified cytochrome b559 from guinea pig macrophages was relipidated with several phospholipid mixtures. Relipidated cytochrome b559 was found capable of NADPH-dependent Superoxide (O2−) production in the absence of the cytosolic components of the NADPH oxidase complex. The rate of O2− generation by cytochrome b559 varied with the type of phospholipid utilized for relipidation, was absolutely dependent on exogenous FAD, and was enhanced by a critical concentration of anionic amphiphile. It is demonstrated that exogenous FAD acts by binding to cytochrome b559. These results provide firm experimental evidence for the proposal that cytochrome b559 comprises the complete electron transporting apparatus of the O2− forming NADPH oxidase and that the cytosolic components function merely as activators

Correlation between multi-drug resistance-associated membrane transport in clonal cancer cells and the cell cycle phase.

Multidrug resistance driven by ABC membrane transporters is one of the major reasons for treatment failure in human malignancy. Some limited evidence has previously been reported on the cell cycle dependence of ABC transporter expression. However, it has never been demonstrated that the functional activity of these transporters correlates with the cell cycle position. Here, we studied the rate of intrinsic ABC transport in different phases of the cell cycle in cultured MCF-7 breast cancer cells. The rate was characterized in terms of the efflux kinetics from cells loaded with an ABC transporter substrate. As averaging the kinetics over a cell population could lead to errors, we studied kinetics of ABC transport at the single-cell level. We found that the rate of ABC transport in MCF-7 cells could be described by Michaelis-Menten kinetics with two classical parameters, V(max) and K(M). Each of these parameters showed similar unimodal distributions with different positions of maxima for cell subpopulations in the 2c and 4c states. Compared to the 2c cells, the 4c cells exhibited greater V(max) values, indicating a higher activity of transport. They also exhibited a greater V(max)/K(M) ratio, indicating a higher efficiency of transport. Our findings suggest that cell cycle-related modulation of MDR may need to be taken into account when designing chemotherapy regimens which include cytostatic agents

Single-Cell-Kinetics Approach to Discover Functionally Distinct Subpopulations within Phenotypically Uniform Populations of Cells

Phenotypically uniform cell populations may contain subpopulations with different activities of enzymes, membrane transporters, and other functional units which can be characterized kinetically. Here we propose the first approach to the discovery of such subpopulations; we term it single-cell approach to discover subpopulations or SCADS for short. SCADS combines microscopy, single-cell kinetic analysis, and population/cluster analysis to discover a functionally distinct subpopulation of cells. In this proof-of-principle work, we used SCADS to search for subpopulations with distinct kinetic patterns of membrane transport in bulk tumor cells (BTCs) and tumor-initiating cells (TICs). We used two classical Michaelis parameters, <i>V</i>_max and <i>K</i>_M, to kinetically characterize the rate of transport. We found that the BTC population was homogeneous with respect to membrane transport. When analyzing TICs, we discovered three main functionally distinct subpopulations: (i) cells with a high rate of transport (high <i>V</i>_max), (ii) cells with high-affinity transporters (low <i>K</i>_M), and (iii) cells with activity and affinity similar to those in BTCs (low <i>V</i>_max and high <i>K</i>_M)

Kinetics of MDR transport in tumor-initiating cells.

Multidrug resistance (MDR) driven by ABC (ATP binding cassette) membrane transporters is one of the major causes of treatment failure in human malignancy. MDR capacity is thought to be unevenly distributed among tumor cells, with higher capacity residing in tumor-initiating cells (TIC) (though opposite finding are occasionally reported). Functional evidence for enhanced MDR of TICs was previously provided using a "side population" assay. This assay estimates MDR capacity by a single parameter - cell's ability to retain fluorescent MDR substrate, so that cells with high MDR capacity ("side population") demonstrate low substrate retention. In the present work MDR in TICs was investigated in greater detail using a kinetic approach, which monitors MDR efflux from single cells. Analysis of kinetic traces obtained allowed for the estimation of both the velocity (V max) and affinity (K M) of MDR transport in single cells. In this way it was shown that activation of MDR in TICs occurs in two ways: through the increase of V max in one fraction of cells, and through decrease of K M in another fraction. In addition, kinetic data showed that heterogeneity of MDR parameters in TICs significantly exceeds that of bulk cells. Potential consequences of these findings for chemotherapy are discussed