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Overexpression and amplification of five genes in a multidrug-resistant Chinese hamster ovary cell line
Abstract:
Multidrug-resistant cells are cross-resistant to a wide range of unrelated drugs, many of which are used in cancer chemotherapy. We constructed a cDNA library from RNA of the multidrug-resistant Chinese hamster ovary cell line CHRC5. By differential screening we isolated cDNAs derived from mRNAs that are overexpressed in this cell line. The cDNAs could be grouped in five classes on the basis of transcript lengths detected in RNA blots. We infer that each class codes for a separate protein. The corresponding genes are amplified 10 or 30 times in CHRC5 DNA, providing an explanation for the constitutive overexpression found in this cell line. Despite differential amplification, the genes may be linked in one large amplicon as indicated by the hybridization analysis of large fragments of CHRC5 DNA separated by pulsed field gradient gel electrophoresis. Therefore, some of these genes might be fortuitously coamplified and not contribute functionally to the resistant phenotype. It is also possible, however, that genes involved in drug resistance are clustered. One of our clones cross-hybridized with the recently described cDNA pCHP1 (J. R. Riordan, K. Deuchars, N. Kartner, N. Alon, J. Trent, and V. Ling, Nature [London] 316:817-819, 1985) encoding part of the 170-kilodalton P-glycoprotein, a protein which is frequently overproduced in multidrug-resistant cells. The nature of the four other genes is still unknown. Sequences of four of the five classes of cDNAs are conserved in mouse and human DNA.
Insights
Researchers identified overexpressed genes in multidrug-resistant cells, finding gene amplification explains resistance. One gene relates to P-glycoprotein, crucial for multidrug resistance in cancer chemotherapy.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Multidrug-resistant cells exhibit cross-resistance to diverse drugs, including those used in cancer chemotherapy.
- Overexpression of specific genes is a known mechanism contributing to multidrug resistance.
Purpose of the Study:
- To identify and characterize genes overexpressed in multidrug-resistant cells.
- To investigate the genetic basis of multidrug resistance in the CHRC5 cell line.
Main Methods:
- Construction of a cDNA library from multidrug-resistant Chinese hamster ovary (CHRC5) cells.
- Differential screening to isolate overexpressed cDNAs.
- RNA blotting and DNA hybridization (including pulsed field gradient gel electrophoresis) to analyze gene expression and amplification.
Main Results:
- Five classes of overexpressed cDNAs were identified, suggesting distinct protein products.
- Genes corresponding to these cDNAs are amplified 10-30 fold in CHRC5 cells, explaining constitutive overexpression.
- One cDNA clone cross-hybridized with pCHP1, encoding part of P-glycoprotein, a known factor in multidrug resistance.
- Evidence suggests these amplified genes may be located within a large amplicon, potentially indicating gene clustering.
Conclusions:
- Gene amplification is a key mechanism underlying multidrug resistance in the studied cell line.
- The identification of P-glycoprotein-related sequences provides insight into drug efflux mechanisms.
- Further research is needed to elucidate the function of the other four identified genes in multidrug resistance.
