Limits...
Kinetics of MDR transport in tumor-initiating cells.

Koshkin V, Yang BB, Krylov SN - PLoS ONE (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, Canada.

ABSTRACT
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.

Show MeSH

Related in: MedlinePlus

Kinetic and morphologic analysis of MCF-7 TICs and bulk cells: (a) typical image of toluidine blue-stained cells; (b) distribution of brightness of stained cells showing discrimination between pale and dark cells; (c, d) histograms of distribution of Michaelis parameters within pale (undifferentiated) cells (gray bars) and dark (differentiated) cells (white bars) demonstrating two ways of MDR activation in TICs; (e) cumulative data summarizing Michaelis parameters in pale (undifferentiated) cells (gray bars) and dark (differentiated) cells (white bars) in 4 independent experiments( total 821 cells, p < 0.01).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815210&req=5

pone-0079222-g003: Kinetic and morphologic analysis of MCF-7 TICs and bulk cells: (a) typical image of toluidine blue-stained cells; (b) distribution of brightness of stained cells showing discrimination between pale and dark cells; (c, d) histograms of distribution of Michaelis parameters within pale (undifferentiated) cells (gray bars) and dark (differentiated) cells (white bars) demonstrating two ways of MDR activation in TICs; (e) cumulative data summarizing Michaelis parameters in pale (undifferentiated) cells (gray bars) and dark (differentiated) cells (white bars) in 4 independent experiments( total 821 cells, p < 0.01).

Mentions: We were also interested in determining the degree of Vmax and KM dispersion within TIC and bulk cell populations. Distributions of Vmax and KM parameters within these populations considerably deviate from normal (Figure 2, d, e; Figure 3, c, d), therefore we chose to characterize Vmax and KM dispersion using the robust coefficient of variation (rCV). rCV is the ratio of interquartile range which spans the central 50% of a data set, to the median value of a data set, which is more appropriate than common CV for description of skewed distributions [37]. Calculations showed that rCVs of Vmax and KM distributions of the TIC population were approximately 2-fold higher than those of the bulk population (data not shown).


Kinetics of MDR transport in tumor-initiating cells.

Koshkin V, Yang BB, Krylov SN - PLoS ONE (2013)

Kinetic and morphologic analysis of MCF-7 TICs and bulk cells: (a) typical image of toluidine blue-stained cells; (b) distribution of brightness of stained cells showing discrimination between pale and dark cells; (c, d) histograms of distribution of Michaelis parameters within pale (undifferentiated) cells (gray bars) and dark (differentiated) cells (white bars) demonstrating two ways of MDR activation in TICs; (e) cumulative data summarizing Michaelis parameters in pale (undifferentiated) cells (gray bars) and dark (differentiated) cells (white bars) in 4 independent experiments( total 821 cells, p < 0.01).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815210&req=5

pone-0079222-g003: Kinetic and morphologic analysis of MCF-7 TICs and bulk cells: (a) typical image of toluidine blue-stained cells; (b) distribution of brightness of stained cells showing discrimination between pale and dark cells; (c, d) histograms of distribution of Michaelis parameters within pale (undifferentiated) cells (gray bars) and dark (differentiated) cells (white bars) demonstrating two ways of MDR activation in TICs; (e) cumulative data summarizing Michaelis parameters in pale (undifferentiated) cells (gray bars) and dark (differentiated) cells (white bars) in 4 independent experiments( total 821 cells, p < 0.01).
Mentions: We were also interested in determining the degree of Vmax and KM dispersion within TIC and bulk cell populations. Distributions of Vmax and KM parameters within these populations considerably deviate from normal (Figure 2, d, e; Figure 3, c, d), therefore we chose to characterize Vmax and KM dispersion using the robust coefficient of variation (rCV). rCV is the ratio of interquartile range which spans the central 50% of a data set, to the median value of a data set, which is more appropriate than common CV for description of skewed distributions [37]. Calculations showed that rCVs of Vmax and KM distributions of the TIC population were approximately 2-fold higher than those of the bulk population (data not shown).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Centre for Research on Biomolecular Interactions, York University, Toronto, Ontario, Canada.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus