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Selective amplification of polymorphic dihydrofolate reductase gene loci in Chinese hamster lung cells
Abstract:
A series of antifolate-resistant Chinese hamster lung sublines that overproduce either a Mr 20,000 or a Mr 21,000 class of dihydrofolate reductase (DHFR; tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3), known to contain amplified DHFR genes, has been analyzed by DNA and RNA transfer techniques. The results suggest that the Mr 20,000 and Mr 21,000 DHFRs are encoded by at least two polymorphic DHFR genes, both of which are expressed in drug-sensitive parental cells. In drug-resistant sublines only one of the two DHFR gene types is amplified, thus accounting for the overproduction of one or the other molecular weight class of DHFR. In addition to the known differences between the DHFRs whose overproduction they direct, these allelic genes differ in restriction endonuclease profiles and in the relative abundances of their multiple mRNA transcripts.
Insights
Chinese hamster lung cells resistant to antifolates overproduce dihydrofolate reductase (DHFR). This resistance stems from amplified DHFR genes, with specific gene types leading to distinct DHFR molecular weights and varying mRNA levels.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Antifolate resistance in cancer therapy is often linked to dihydrofolate reductase (DHFR) gene amplification.
- Chinese hamster lung cell lines provide a model for studying drug resistance mechanisms.
- DHFR plays a crucial role in nucleotide synthesis and is a target for antifolate drugs.
Purpose of the Study:
- To investigate the genetic basis of differential DHFR overproduction in antifolate-resistant Chinese hamster lung cell sublines.
- To determine if distinct DHFR gene alleles are responsible for the observed variations in DHFR molecular weight and expression.
- To analyze the correlation between gene amplification, mRNA abundance, and protein expression in drug-resistant cells.
Main Methods:
- Analysis of DNA and RNA transfer techniques in antifolate-resistant and sensitive cell lines.
- Characterization of dihydrofolate reductase (DHFR) gene amplification patterns.
- Quantification of DHFR mRNA transcripts using molecular hybridization techniques.
- Assessment of DHFR protein molecular weight classes.
Main Results:
- Antifolate-resistant Chinese hamster lung sublines exhibit overproduction of either Mr 20,000 or Mr 21,000 DHFR.
- At least two polymorphic DHFR genes, both expressed in drug-sensitive cells, encode these DHFR variants.
- Drug-resistant sublines show amplification of only one DHFR gene type, correlating with the overproduction of a specific DHFR molecular weight class.
- Allelic DHFR genes differ in restriction endonuclease profiles and mRNA transcript abundance.
Conclusions:
- The differential overproduction of DHFR molecular weight classes in antifolate-resistant cells is attributed to the selective amplification of specific allelic DHFR genes.
- Polymorphic DHFR genes contribute to varying resistance levels and expression patterns.
- Understanding these genetic variations is crucial for developing targeted antifolate therapies.