Genetic and biochemical characteristics of three different types of mutants of mammalian cells affected in adenosine kinase.
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Adenosine↗
Biomedical subjects
K D Mehta
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Novel mutants of CHO cells resistant to adenosine analogs and containing biochemically altered form of adenosine kinase in cell extracts.
Stable mutants which are approximately five- and eightfold resistant to an inosine analog, formycin B (Fomr) have been selected in a single-step from Chinese hamster ovary cells at a frequency of approximately 10(-6). Cross-resistance studies with these mutants show that the Fomr mutants exhibit increased resistance to all adenosine analogs (N-and C-nucleosides) examined and, in accordance with their cross-resistance pattern, the mutants exhibited decreased cellular uptake and phosphorylation of formycin B and various adenosine analogs. In cell hybrids formed with sensitive cells, the drug-resistant phenotype of these mutants behaved recessively. However, unlike mutants resistant to adenosine analogs that have been obtained previously, which contain no measurable activity of adenosine kinase (AK) in cell extracts, the two Fomr mutants studied contained about 60 and 110% of the enzyme activity (compared to the parental cells) in their cell extracts. Biochemical studies with AK from the mutant cells show that in comparison to the wild-type enzyme, the mutant enzymes required much higher concentrations of the adenosine analog N7-(delta 2-isopentenyl) formycin A for similar inhibition of [3H]adenosine phosphorylation. These results indicate that AK from the Fomr mutants has lower affinity for phosphorylation of adenosine analogs in comparison to the enzyme from the parental cells. The genetic lesion in the Fomr mutants may thus be directly affecting the structural gene for AK.
Adenosine↗
Immunological studies with different classes of mutants affected at the adenosine kinase locus in CHO cells.
Adenosine kinase (AK) from CHO cells has been purified to homogeneity and specific antibodies to it have been raised in rabbits. Using this antibody, the presence of a specific cross-reacting protein (CRP) in cell extracts of different classes of mutants resistant to purine nucleoside analogs which are affected in AK has been investigated by the immunoblotting technique. Results of our studies show that 31 of the 32 independently selected class A AK- mutants (obtained at high frequency in presence of adenosine analogs toyocamycin, tubercidin, 6-methylmercaptopurine riboside, or pyrazofurin and containing no measurable activity of AK in cell extracts) contained similar amounts of a specific CRP as seen in the parental AK+ cells. The CRP in the parental and different mutant cell lines has the same relative molecular mass as purified AK. Similar results were obtained with two mutants each of the class B and C type (selected in presence of C-nucleosides formycin A and formycin B), which are also affected in AK but show novel properties. The presence of equivalent amounts of the CRP in the vast majority of the class A mutants strongly indicates that the high frequency of those mutants in CHO cells is not a result of an epigenetic or deletion type of event, but that such mutants may contain missense types of mutations at a presumed "mutational hot spot" within the structural gene for adenosine kinase.
Adenosine Kinase↗
Involvement of adenosine kinase in the phosphorylation of formycin B in CHO cells.
In Chinese hamster ovary cells, [3H]formycin B is metabolized into formycin B-5'-monophosphate, formycin A-5'-monophosphate and higher phosphorylated derivatives of formycin A which are incorporated into RNA. Mutants of CHO cells independently selected for resistance to various adenosine analogs viz. toyocamycin, tubercidin, 6-methylmercaptopurine riboside, which contain no detectable activity of adenosine kinase (AK) in cell extracts, all exhibited between 2- to 3-fold increased resistance to formycin B. Formycin B-resistant mutants of CHO cells are also affected in AK, as indicated by the absence of AK activity in cell extracts. Both types of AK- mutants showed reduced uptake and phosphorylation of [3H]formycin B in comparison to the parental (AK+) cells. In addition, toxicity of formycin B towards CHO cells was reduced in presence of adenosine in a concentration dependent manner. These observations strongly indicate that in CHO cells, formycin B is phosphorylated via AK and that like other nucleoside analogs its phosphorylation may be essential for the drugs cellular toxicity.
Adenosine↗
Chinese hamster ovary cell mutants specifically affected in the phosphorylation of C-purine nucleosides.
In Chinese hamster ovary cells, two different types of mutants resistant to purine nucleoside analogs have been isolated. One type of mutants selected in presence of C-nucleosides formycin A and formycin B (FomR mutants) exhibited a high degree of cross resistance to different C-nucleosides but showed very slight to no cross resistance to various N-nucleosides. In contrast, mutants selected in presence of toyocamycin (Toyr mutants) exhibited a high degree of cross resistance to all C- and N-adenosine analogs. Studies on the cellular uptake and phosphorylation of [3H]adenosine, [3H]tubercidin, and [3H]formycin A show that, unlike the Toyr mutants which show reduced phosphorylation of all three compounds, the FomR mutants show reduced cellular phosphorylation of only [3H]formycin A. It is suggested that the genetic lesion in the FomR mutants affects adenosine kinase in a novel manner that specifically affects the phosphorylation of C-purine nucleosides.
Adenosine↗
Genetic and biochemical studies on mutants of CHO cells resistant to 7-deazapurine nucleosides: differences in the mechanisms of action of toyocamycin and tubercidin.
From mutants of Chinese hamster ovary cells which are resistant to toyocamycin and tubercidin, second-step mutants which exhibit a further 8- to 9-fold increase in resistance to toyocamycin (ToyrII mutants) but no change in resistance to tubercidin have been isolated. The ToyrII mutants are similar to the first-step mutants in their levels of adenosine kinase activity (congruent to 1%), as well as cellular uptake and phosphorylation of adenosine and its analogs. The increased resistance of the ToyrII mutants to toyocamycin but not to tubercidin provides strong evidence that the mechanism of cellular toxicity of these two analogs is different from each other and suggests that these mutants may be affected in a cellular component which is specifically involved in the toxicity of toyocamycin. The ToyrII mutants also exhibit increased resistance to sangivamycin and the tricyclic nucleoside pentaaza-acenaphthylene riboside (TCN, NSC 154020) indicating that the mechanisms of cellular toxicity of these two analogs may be similar to that of toyocamycin.
Animals↗
Formycin B-resistant mutants of Chinese hamster ovary cells: novel genetic and biochemical phenotype affecting adenosine kinase.
Stable mutants which are approximately three- and eightfold resistant to the pyrazolopyrimidine nucleosides formycin A and formycin B (FomR) have been selected in a single step from mutagenized Chinese hamster ovary cells. In cell extracts, the two FomR mutants which were examined were both found to contain no measurable activity of the enzyme adenosine kinase (AK). However, cross-resistance studies with other adenosine analogs such as toyocamycin and tubercidin show that these mutants are distinct from toyocamycin or tubercidin resistant (Toyr) mutants which also contain no measurable AK activity in cell extracts. Studies on the uptake and incorporation of [3H]adenosine and [3H]tubercidin by various mutants and parental cell lines show that unlike the Toyr mutants, which are severely deficient in the phosphorylation of these compounds, the FomR mutants possess nearly normal capacity to phosphorylate these compounds and incorporate them into cellular macromolecules. These results suggest that the FomR mutants contain normal levels of AK activity in vivo. In cell hybrids formed between FomR X FomS cells and FomR X Toyr cells, the formycin-resistant phenotype of both of the FomR mutants behaved codominantly. However, the extracts from these hybrid cells contained either congruent to 50% (FomR X FomS) or no measurable (FomR X Toyr) AK activity, indicating that the lesion in these mutants neither suppresses the wild-type AK activity nor complements the AK deficiency of the Toyr mutants. The presence of AK activity in the FomR mutants in vivo, but not in their cell extracts, along with the codominant behavior of the mutants in hybrids, indicates that the lesions in the FomR mutant are of a novel nature. It is suggested that the genetic lesion in these mutants affects AK activity indirectly and that it may involve an essential cellular function which exists in a complex form with AK. Some implications of these results regarding the mechanism of action of formycin B are discussed.
Adenosine↗