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Increased mdr gene expression and decreased drug accumulation in multidrug-resistant human melanoma cells
J F Lemontt1, M Azzaria, P Gros
1Integrated Genetics, Inc., Framingham, Massachusetts 01701.
Abstract:
Multidrug-resistant clones of a drug-sensitive human malignant melanoma cell line were isolated by single-step selection in culture medium containing either vincristine (4.5 ng/ml or 7.5 ng/ml), vinblastine (3 ng/ml), or colchicine (8 ng/ml). This protocol yielded primary colonies showing relatively low (4- to 24-fold) levels of drug resistance. These clones exhibit the classical multidrug resistance (MDR) phenotype, being cross-resistant to Vinca alkaloids, anthracyclines, colchicine, and actinomycin D. The appearance of an MDR phenotype in these cells was linked to a decreased accumulation and increased efflux of the drug [3H]vinblastine when compared to the drug-sensitive melanoma cell line. This increased drug efflux was dependent on the presence of cellular ATP and could be reduced by treatment of the cells with rotenone and deoxyglucose. A partial human mdr complementary DNA clone was used to monitor the degree of amplification and the level of transcription of this gene in the cloned lines. All 5 MDR sublines expressed increased levels of the specific 4.5-kilobase mdr mRNA, but did not show mdr gene amplification. Our results indicate that relatively low levels of drug resistance, similar to those observed clinically and in experimental xenografts, can be achieved by single-step drug selection and result from increased expression of at least one member of the mdr gene family.
Insights
Researchers developed multidrug-resistant (MDR) melanoma cells using single-step selection. These cells showed increased mdr gene expression, not amplification, leading to low-level drug resistance relevant to clinical settings.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Multidrug resistance (MDR) is a significant challenge in cancer chemotherapy.
- Understanding the mechanisms of MDR development is crucial for improving treatment efficacy.
Purpose of the Study:
- To generate and characterize multidrug-resistant human malignant melanoma cell lines.
- To investigate the molecular mechanisms underlying acquired low-level drug resistance.
Main Methods:
- Single-step drug selection of a sensitive melanoma cell line using vincristine, vinblastine, or colchicine.
- Phenotypic characterization of drug resistance and cross-resistance patterns.
- Assessment of drug accumulation and efflux using [3H]vinblastine.
- Analysis of mdr gene expression and amplification using a human mdr cDNA probe.
Main Results:
- Isolated MDR clones exhibited cross-resistance to Vinca alkaloids, anthracyclines, colchicine, and actinomycin D.
- MDR phenotype correlated with decreased intracellular drug accumulation and increased ATP-dependent drug efflux.
- All MDR sublines showed increased mdr mRNA levels without mdr gene amplification.
- Achieved low-level (4- to 24-fold) drug resistance, mimicking clinical and xenograft observations.
Conclusions:
- Single-step drug selection can efficiently generate MDR melanoma cells with clinically relevant resistance levels.
- Acquired low-level MDR in this model is primarily driven by increased expression of mdr gene family members, not gene amplification.
- These findings provide insights into the early mechanisms of MDR development in cancer therapy.