Related Experiment Videos
Adenosine deaminase gene amplification in deoxycoformycin-resistant mammalian cells
Abstract:
Deoxycoformycin (dCF)-resistant mutants of rat hepatoma, mouse LMTK-, and Chinese hamster ovary (CHO) cells have been isolated and shown to overproduce adenosine deaminase (ADA). The overproduction of ADA was found to be due to ADA-gene amplification in rat and mouse cells but not in CHO cells. Deoxycoformycin-resistant rat hepatoma cells have large HSRs (homogeneously staining regions), mouse cells carry DMs (Double minutes), and CHO cells do not appear to have any gross chromosomal anomalies. When dCF-resistant rat hepatoma and mouse cells are selected by increasing the concentration of the inhibitor in small increments, there is a good correlation between the increase in ADA gene copy number and the increase in the level of expression of ADA, suggesting that all of the amplified genes are equally active in the expression of ADA.
Insights
Deoxycoformycin-resistant cells overproduce adenosine deaminase (ADA) due to gene amplification in rat and mouse cells. Amplified ADA genes correlate with increased enzyme expression, indicating uniform activity.
Area of Science:
- Molecular Biology
- Genetics
- Cell Biology
Background:
- Adenosine deaminase (ADA) is a crucial enzyme in purine metabolism.
- Drug resistance can arise from alterations in gene expression and copy number.
- Deoxycoformycin (dCF) is a potent inhibitor of ADA.
Purpose of the Study:
- To investigate the mechanisms of dCF resistance in mammalian cells.
- To determine the genetic basis for ADA overproduction in resistant cell lines.
- To correlate gene amplification with enzyme activity levels.
Main Methods:
- Isolation and characterization of dCF-resistant cell mutants (rat hepatoma, mouse LMTK-, CHO).
- Analysis of ADA gene copy number and expression levels.
- Cytogenetic analysis to identify chromosomal abnormalities (HSRs, DMs).
Main Results:
- dCF-resistant cells exhibited significant overproduction of ADA.
- ADA gene amplification was identified as the cause in rat and mouse cells, but not CHO cells.
- Rat cells showed homogeneously staining regions (HSRs), mouse cells had double minutes (DMs), and CHO cells lacked gross chromosomal changes.
- A strong correlation was observed between increased ADA gene copy number and elevated ADA expression levels in resistant cells.
Conclusions:
- ADA gene amplification is a primary mechanism for dCF resistance in specific mammalian cell types.
- The extent of gene amplification directly influences ADA enzyme expression.
- Different chromosomal anomalies (HSRs, DMs) are associated with gene amplification in distinct cell lines.