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The human multidrug resistance (mdr1) gene. cDNA cloning and transcription initiation
Abstract:
Multidrug resistance in human KB carcinoma cells selected for resistance to colchicine, vinblastine, or adriamycin results from overexpression, and frequently amplification, of a specific gene (mdr1). Overlapping cDNA clones representing a complete 4.7-kilobase mdr1 transcript have been obtained from multidrug-resistant KB cells. Primer extension and S1 nuclease protection experiments show that two transcripts initiate 136 and 140 bases upstream from the first ATG codon in all human multidrug-resistant cell lines. The mdr1 gene is expressed in human normal kidney cells and HepG2 liver cells as a poly(A)+ RNA which starts from the same sites. Less prominent transcripts were found to initiate 155-180 bases upstream from the first ATG codon in vinblastine- or adriamycin-selected cell lines and 480-630 bases upstream in colchicine-selected cell lines. Southern hybridization analyses with different portions of a full-length cDNA indicate that the human mdr1 gene encompasses at least 70 kilobases of DNA amplified in all highly multidrug-resistant cell lines.
Insights
Multidrug resistance in cancer cells stems from the mdr1 gene. This gene
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Multidrug resistance (MDR) is a significant challenge in cancer chemotherapy.
- Overexpression and amplification of the multidrug resistance gene (mdr1) are key mechanisms in MDR.
- KB carcinoma cells selected for resistance to colchicine, vinblastine, or adriamycin exhibit MDR.
Purpose of the Study:
- To characterize the mdr1 gene transcript and its expression in multidrug-resistant human cells.
- To investigate the transcriptional start sites and gene structure of mdr1.
- To understand the genetic basis of multidrug resistance in human KB carcinoma cells.
Main Methods:
- Obtained overlapping cDNA clones for the 4.7-kilobase mdr1 transcript.
- Utilized primer extension and S1 nuclease protection assays to determine transcript initiation sites.
- Performed Southern hybridization analyses to assess mdr1 gene amplification and size.
Main Results:
- Identified two primary mdr1 transcripts initiating 136 and 140 bases upstream of the ATG codon in all MDR cell lines.
- Confirmed mdr1 gene expression in normal human kidney and HepG2 liver cells from the same start sites.
- Detected less prominent transcripts with alternative start sites in specific drug-selected cell lines.
- Southern analysis indicated the human mdr1 gene spans at least 70 kilobases and is amplified in MDR cells.
Conclusions:
- The mdr1 gene is amplified and overexpressed in multidrug-resistant human KB carcinoma cells.
- Specific transcriptional start sites for mdr1 are conserved across different MDR cell lines and also present in normal cells.
- Alternative transcription initiation contributes to the complex expression patterns of mdr1 in response to different chemotherapeutic agents.