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Purine uptake by azaguanine-resistant Chinese hamster cells
Abstract:
In this study the resistance of a number of lines of Chinese hamster ovary cells to azaguanine is examined. Those which are drug resistant by virtue of a deficiency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) fail to take up any exogenous hypoxanthine or azaguanine. A second class of drug resistant cells which grow in the reverse selective HAT medium and have levels of HPRT in the range of the wild type parent line take up these purines at lower rates than the nonresistant cells and incorporate smaller amounts of them into trichloracetic acidinsoluble constituents. The results suggest that their basis for resistance resides in lowered incorporation of azaguanine into DNA and RNA, possibly due to a mofified HPRT molecule which accepts hypoxanthine, but not azaguanine as a substrate.
Insights
Chinese hamster ovary cells resistant to azaguanine exhibit two distinct mechanisms. Some lack hypoxanthine-guanine phosphoribosyltransferase (HPRT), while others show reduced purine uptake and incorporation due to a modified HPRT.
Area of Science:
- Cell Biology
- Biochemistry
- Genetics
Background:
- Azaguanine is a purine analog used to select for drug-resistant cells.
- Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a key enzyme in purine salvage pathways.
- Chinese hamster ovary (CHO) cells are a common model system in toxicology and genetics.
Purpose of the Study:
- To investigate the mechanisms of azaguanine resistance in CHO cells.
- To differentiate between HPRT-deficient and other forms of resistance.
- To understand the role of purine uptake and incorporation in drug resistance.
Main Methods:
- Culturing CHO cell lines with varying azaguanine resistance levels.
- Measuring the uptake of radiolabeled hypoxanthine and azaguanine.
- Assessing the incorporation of these purines into trichloroacetic acid-insoluble cellular components (DNA and RNA).
Main Results:
- Two classes of azaguanine-resistant CHO cells were identified.
- Class 1: Cells deficient in HPRT showed no uptake of exogenous hypoxanthine or azaguanine.
- Class 2: Cells with near-wild-type HPRT levels exhibited reduced purine uptake and incorporation into nucleic acids.
Conclusions:
- Azaguanine resistance in CHO cells can arise from HPRT deficiency or altered purine metabolism.
- A modified HPRT enzyme, functional for hypoxanthine but not azaguanine, may underlie the second resistance mechanism.
- Reduced incorporation of azaguanine into DNA and RNA is a key feature of this modified HPRT-mediated resistance.