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Molecular/cytogenetic alterations accompanying the development of multidrug resistance in the J774.2 murine cell line
M L Slovak1, L Lothstein, S B Horwitz
1Department of Radiation Oncology, Arizona Cancer Center, University of Arizona, Tucson 85724.
Abstract:
Mouse macrophage-like J774.2 cells were selected for resistance to colchicine and examined by molecular/cytogenetic analysis to determine whether the acquisition of the multidrug resistant (mdr) phenotype was associated with specific chromosomal rearrangements. Cytogenetic studies of the J774.2 parental and two colchicine-resistant (CLCR) sublines--J7.Cl-30 (770-fold CLCR) and J7.Cl-100 (2500-fold CLCR)--demonstrated specific numeric and structural karyotypic alterations accompanying the emergence of mdr. The parental cells demonstrated a modal chromosome number of 63, while the modal number of the J7.Cl-30 subline was 53. The most striking difference between the parental and J7.Cl-30 subline was the presence of an average of 60 double minutes (DMs) per cell in the CLCR cells. The 2500-fold resistant J7.Cl-100 subline displayed a modal number of 50, which included structural rearrangements involving chromosomes 2 and 7 and concomitant replacement of DMs by a homogeneously staining region (HSR). Southern blotting analysis demonstrated a approximately 35-fold amplification of P-glycoprotein homologous sequences in the J7.Cl-30 subline and approximately 70-fold amplification in the J7.Cl-100 subline. Chromosomal in situ hybridization localized the amplified P-glycoprotein sequences to DMs (J7.Cl-30) and the HSR (J7.Cl-100) in these CLCR sublines. Our results suggest that CLCR in J774.2 cells results from overexpression of P-glycoprotein via gene amplification which was accompanied by chromosomal evolution from DMs to an HSR.
Insights
Mouse cells developed multidrug resistance (mdr) through P-glycoprotein gene amplification. This resistance was linked to chromosomal changes, evolving from double minutes (DMs) to homogeneously staining regions (HSRs).
Area of Science:
- Cell Biology
- Genetics
- Molecular Biology
Background:
- Multidrug resistance (mdr) is a significant challenge in cancer therapy.
- The P-glycoprotein (P-gp) is a key transporter involved in extruding drugs from cells.
- Chromosomal abnormalities are often associated with the development of drug resistance phenotypes.
Purpose of the Study:
- To investigate the chromosomal basis of colchicine resistance (CLCR) in mouse macrophage-like J774.2 cells.
- To determine the association between the acquisition of the mdr phenotype and specific chromosomal rearrangements.
- To analyze the role of P-glycoprotein gene amplification in CLCR.
Main Methods:
- Cytogenetic analysis of parental and colchicine-resistant (CLCR) J774.2 cell sublines.
- Molecular analysis including Southern blotting to assess gene amplification.
- Chromosomal in situ hybridization (ISH) to localize amplified gene sequences.
Main Results:
- CLCR sublines exhibited significant numeric and structural karyotypic alterations.
- Colchicine-resistant cells showed an increase in double minutes (DMs) or homogeneously staining regions (HSRs).
- P-glycoprotein gene amplification correlated with the level of CLCR, with amplified sequences localized to DMs and HSRs.
Conclusions:
- Colchicine resistance in J774.2 cells arises from P-glycoprotein overexpression due to gene amplification.
- The evolution from DMs to HSRs represents a mechanism of chromosomal adaptation during the development of drug resistance.
- These findings provide insights into the genetic and chromosomal mechanisms underlying multidrug resistance.