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Implementation of In Vitro Drug Resistance Assays: Maximizing the Potential for Uncovering Clinically Relevant Resistance Mechanisms
Published on: December 9, 2015
Multiple resistance mechanisms in Chinese hamster cells resistant to 9-hydroxyellipticine
A K Larsen1, A Jacquemin-Sablon
1Laboratoire de Pharmacologie Moléculaire, UA 147 CNRS, Villejuif, France.
Abstract:
We have previously shown that Chinese hamster lung cells resistant to 9-hydroxyellipticine, DC-3F/9-OH-E, display multiple phenotypical alterations including cross-resistance to a variety of drugs as well as loss of tumorigenicity. We now analyze a DC-3F/9-OH-E subline that has been maintained for a prolonged period of time in drug-free medium in order to clarify the relationships between the various phenotypic traits. The absence of selection resulted in a partial recovery of the ability to form colonies in soft agar as well as of the tumorigenicity in nude mice. In contrast, no change was observed with respect to population-doubling time. Our results also show that the resistance to 9-hydroxyellipticine, which is associated with an altered topoisomerase II activity, is stable in the absence of drug for more than 1 year. In contrast, the cross-resistance to doxorubicin is partially reversible and the cross-resistance to vincristine is totally reversible in the absence of selection. The cross-resistance to vincristine and doxorubicin is accompanied by a decreased drug uptake. Northern blot analysis shows that the multidrug resistance-associated Mr 170,000-180,000 glycoprotein is overexpressed in the DC-3F/9-OH-E cells and that the overexpression is lost in the absence of selection. We conclude that (a) the DC-3F/9-OH-E cells exhibit multiple mechanisms of resistance which can be dissociated, (b) the tumorigenicity and the altered topoisomerase activity are independent biochemical events whereas the oncogenic potential appears to follow the expression of the multidrug resistance, and (c) the multidrug resistance phenotype may be induced by a drug which is not itself recognized by the multidrug resistance mechanism such as 9-hydroxyellipticine.
Insights
Chinese hamster lung cells resistant to 9-hydroxyellipticine (9-OH-E) showed reversible multidrug resistance and loss of tumorigenicity. Resistance to 9-OH-E and altered topoisomerase II activity remained stable, indicating independent mechanisms.
Area of Science:
- Cell Biology
- Molecular Pharmacology
- Cancer Research
Background:
- Chinese hamster lung cells (DC-3F/9-OH-E) resistant to 9-hydroxyellipticine (9-OH-E) exhibit pleiotropic phenotypic alterations, including multidrug resistance and reduced tumorigenicity.
- The relationship between these traits and the underlying molecular mechanisms remains incompletely understood.
Purpose of the Study:
- To investigate the stability of various phenotypic traits in DC-3F/9-OH-E cells after prolonged cultivation in drug-free medium.
- To elucidate the interdependencies between drug resistance, topoisomerase II activity, drug uptake, multidrug resistance-associated glycoprotein expression, and tumorigenicity.
Main Methods:
- Cultivation of DC-3F/9-OH-E cells in drug-free medium.
- Assessment of colony formation in soft agar and tumorigenicity in nude mice.
- Measurement of population-doubling time.
- Assay of topoisomerase II activity.
- Determination of drug uptake for doxorubicin and vincristine.
- Northern blot analysis for multidrug resistance-associated glycoprotein (P-glycoprotein) expression.
Main Results:
- Prolonged drug withdrawal led to partial recovery of anchorage-independent growth and tumorigenicity, but not population-doubling time.
- 9-OH-E resistance and altered topoisomerase II activity were stable, while cross-resistance to doxorubicin and vincristine was partially and fully reversible, respectively.
- Reversible cross-resistance correlated with decreased drug uptake and loss of P-glycoprotein overexpression.
- Tumorigenicity loss was independent of stable 9-OH-E resistance and altered topoisomerase II activity.
Conclusions:
- The DC-3F/9-OH-E cell line exhibits multiple, dissociable resistance mechanisms.
- Tumorigenicity and altered topoisomerase II activity are independent events; oncogenic potential correlates with multidrug resistance expression.
- 9-OH-E can induce a multidrug resistance phenotype without being a substrate for the multidrug resistance mechanism.
