GENETIC AND BIOCHEMICAL STUDIES WITH CHINESE HAMSTER OVARY CELL MUTANTS RESISTANT TO THE PURINE NUCLEOSIDE ANALOGS: TOYOCAMYCIN, FORMYCIN A AND FORMYCIN B

摘要

The aim of this study was to investigate the molecular mechanisms of cellular resistance and toxicity to the purine nucleoside analogs toyocamycin, formycin A and formycin B in Chinese hamster ovary (CHO) cells by using genetic, biochemical and immunological approaches.;Three different types of mutants affected in the enzyme adenosine kinase (AK) have been isolated using the above nucleoside analogs as selective agents. First type of mutants (designated class A) obtained using toyocamycin exhibit high degree of cross-resistance and greatly reduced phosphorylation of both C- as well as N-adenosine analogs. These mutants contain no measurable activity of AK in cell extracts. The second type of mutants (class B) obtained using formycin A exhibit increased cross-resistance and reduced cellular phosphorylation of only C-adenosine analogs. Cell extracts from these mutants also contain no measurable activity of AK. The third type of mutants (class C) selected using formycin B, exhibit a low degree of cross-resistance and reduced phosphorylation of both C- as well as N-adenosine analogs. Cell extracts from these mutants contain between 50-100% of AK activity, but its affinity towards adenosine analogs was found to be altered in comparison to the wild-type enzyme. These studies show that the most common mechanism for the development of cellular resistance to the above nucleoside analogs results from an alteration in the enzyme AK which is required for their phosphorylation. The above mutants have also been used to show the involvement of AK in the phosphorylation of formycin B in CHO cells.;To gain further insight into the nature of the genetic lesions involved in the different types of mutants affected in AK, this enzyme from CHO cells was purified to homogeneity. Antibodies which specifically cross-reacts with AK have been raised. Immunoblot analyses of the above mutants showed the presence of a cross-reacting protein of same relative molecular mass and in an amount similar to that present in the parental cells. These results strongly suggest that the different types of mutants contain a missense type of genetic alteration in the structural gene of AK. Furthermore, two-dimensional gel electrophoretic pattern of total cellular proteins from different mutants support the above inference.