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mdr1/P-glycoprotein gene segments analyzed from various human leukemic cell lines exhibiting different multidrug
1Physiologisch-chemisches Institut, Universität Tübingen, Federal Republic of Germany.
Abstract:
Three high-level multidrug-resistant sublines of the human T-lymphoblastoid cell line CCRF-CEM were selected independently with either actinomycin D, vincristine or adriamycin. They exhibited distinct quantitative differences of cross-resistance profiles, and showed amplification and marked expression of the mdrl/P-glycoprotein gene. DNA and RNA were prepared from the cell lines, and additionally from three cell samples of patients suffering from acute lymphatic leukemia. Applying the polymerase chain reaction (PCR) for amplification, we cloned and sequenced from these sources segments of the mdrl/P-glycoprotein gene around the codon 185 which codes for an amino acid residue possibly influencing the drug binding function of the P-glycoprotein. Altogether, only 2 single nucleotide differences in an intron were found in 2 out of 40 recombinants each harboring a 209 bp genomic or a 269 bp cDNA fragment of the mdrl/P-glycoprotein gene. Our result does not support the idea of clustered point mutations in this segment of the P-glycoprotein gene as a cause of different multidrug resistance profiles. We additionally examined another segment of the P-glycoprotein gene in its second half, essentially delivering the same negative result, though.
Insights
Multidrug resistance in cancer cells is not caused by clustered point mutations in the MDR1/P-glycoprotein gene. Genetic analysis of leukemia cell lines and patient samples revealed no significant mutations in key gene segments.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- Multidrug resistance (MDR) is a major challenge in cancer chemotherapy.
- The MDR1 gene, encoding P-glycoprotein, is implicated in pumping drugs out of cells.
- Distinct MDR profiles suggest underlying genetic variations.
Purpose of the Study:
- To investigate the role of point mutations in the MDR1/P-glycoprotein gene in conferring distinct multidrug resistance profiles.
- To analyze specific gene segments in cell lines and patient samples for mutations.
Main Methods:
- Selection of multidrug-resistant CCRF-CEM cell sublines (actinomycin D, vincristine, adriamycin).
- DNA and RNA preparation from cell lines and acute lymphatic leukemia patient samples.
- Polymerase chain reaction (PCR) amplification, cloning, and sequencing of MDR1/P-glycoprotein gene segments (around codon 185 and second half).
Main Results:
- Amplification and high expression of the MDR1/P-glycoprotein gene were observed in resistant cell lines.
- Analysis of 40 recombinants revealed only 2 single nucleotide differences in an intron, not in coding regions.
- Examination of another gene segment yielded similar negative results for mutations.
Conclusions:
- The findings do not support the hypothesis that clustered point mutations in the analyzed MDR1/P-glycoprotein gene segments cause diverse multidrug resistance profiles.
- Alternative mechanisms likely contribute to the distinct resistance phenotypes observed in cancer cells.