8-Azaguanine cell resistance

TABLE 5. Frequency of Spontaneous and Chemically Induced Mutations in Chinese Hamster Cells Resistant to Various Levels of 8-Azaguanine ... 

We have studied the influence of the two inhibitors of the enz3mie poly(ADP-ribose) polymerase, benzamide (BZA) and nicotinamide (NA) on N-methyl-N -nitro-N-nitroso-guanidine (MNNG), X-ray, and UV-induced killing and 8-azaguanine (AZ) resistant mutation in V79 Chinese hamster cells. 

Sirianni SR, Huang CC. 1980. Comparison of induction of sister chromatid exchange, 8-azaguanine- and ouabain-resistant mutants by cyclophosphamide, ifosfamide and l-(pyridyl-3)-3,3-dimethyltriazene in Chinese hamster cells cultured in diffusion chambers in mice. Carcinogenesis 1 353-355. 

One of the first demonstrations that acquired resistance could be circumvented was the inhibition of 5. faecalis resistant to 8-azaguanine by 8-azaxanthine. Thus, cells that possess xanthylic acid phosphoribosyltransferase activity could form 8-azaxanthylic acid, which was then converted to 8-azaguanylic acid and incorporated in nucleic acids as such [98]. 

Chinese hamster cells 8-Azaguanine and ovarian resistance... 

Mutations to loss of activity of this enzyme cause resistance to the guanine analogues 8-azaguanine and 6-thioguanine. Thus, wild-type HPRT " cells can be selected against by incorporation of one of these analogues. 

Lehmann, and C.M. Coleila. A comparison of the 8-azaguanine and ouabain-resistance systems for the selection of induced mutant Chinese hamster cells. Mutat. Res. 33 261-278, 1975. 

Azaguanine-resistant avian sarcoma virus-infected cells were less tu-morigenic than nonresistant cells in hamsters. Incubation of cells (infected with adenoviruses in tissue culture) with 8-azaguanine (10 M) inhibited reproduction of the virus. At this concentration, the drug was not toxic for healthy mammalian cells. ... 

Cells are cultured in RPMI1640 with glutamine and 10% FBS. The cells are resistant to 10 mM 8-azaguanine and die in the presence of HAT medium. 

FO, which is a Sp2 + Sp2 hybrid, may require frequent recloning Y3 is a rat myeloma, AI2 a human myeloma, the others are from mouse. Drug resistance 8-Ag and 6-Tg 8-azaguanine and 6-thioguanine (cells lack hypoxanthine phosphoribosyl transferase) BUdR S-bromo-2-deoxyuridine (cells lack thymidine kinase). 

Revertants of myeloma cells may occasionally occur. It is good practice to culture the cells 2 or 3 times a year in the presence of the drug to which the cell line is resistant (e.g. 2xlO M 8-azaguanine). 

Human neoplasm cells in culture show a different type of resistance to 6-mercaptopurine from that cited above, in that they delete the enzyme responsible for converting this pro-drug to the therapeutic nucleotide (6-thioinosine 5 -phosphate) (Brockman, 1963). The deleted enzyme is inosine 5 -phosphate pyrophosphorylase. Similarly, resistance to 8-azaguanine is accompanied by loss of the enzyme guanosine 5 -phosphate pyrophosphorylase in human epidermoid carcinoma cells (Brockman et al., 1961). Resistance to 5-fluoro-uracil also depends on the uneconomic cell s ceasing to convert this pro-drug to the nucleotide. 

X-linked recessive mutations would readily be detected in XY or XO cells. The azaguanine-resistant mutants in the hamster cells which we have studied (Chu et al.y 19696) are hypothesized to be X-linked. The particular cell line, V79, was originally derived from the lung tissue of a male animal (Ford and Yerganian, 1958). 

TABLE 1. Growth Response of Chinese Hamster Cells Sensitive or Resistant to 8-Azaguanine ... 

TABLE 2. Distribution of Azaguanine-Resistant Mutants in Chinese Hamster Cells According to Their Spontaneous or Induced Revertibility to Drug Sensitivity... 

TABLE 4. The Relationship Between Inoculum Size and the Frequency of Mutation (from Azaguanine Sensitivity to Resistance) in Chinese Hamster Cells in Culture ... 

Spontaneous mutations in mammalian cell cultures lead to an accumulation of mutant cells in the population, provided that the mutant cells are not at a selective disadvantage under the in vitro conditions. The spontaneous mutation rate per cell per division at different loci can be estimated by various methods, including the Po method in the fluctuation test (Luria and Delbriick, 1943). For example, in hamster cells the spontaneous forward mutation rate to azaguanine resistance was estimated as 1.5 x 10 per cell per generation the spontaneous reverse mutation rate from glutamine auxotrophy to prototrophy was estimated as 1.4 X 10 per cell per generation (Chu et al.y 19696). 

First, it is possible that a certain portion of the newly arisen mutants do not survive and would remain undetected. The reasons for this excessive killing of mutants and thus reduction in mutation frequency may be imperfect selective media, a competition between the mutant and nonmutant cells, or many other conditions. Nonetheless, this reduction in the survival of mutant cells, if it occurred, may be estimated by a reconstruction experiment. For example, one can mix a known number, such as 100, of previously isolated mutant cells with a large number of mutagen-treated or untreated wild-type cells in a plate containing selective medium. The same number of the mutant cells alone is separately plated as a control. We have performed such experiments using azaguanine-resistant or -sensitive hamster cells as... 

For mutation study, about 10 cells were treated and allowed 7-9 days of expression, with 3-4 subcultures. Cells were then replated at concentration of 5 x 10 per 100 mm Coming dishes in selection medium containing 3 jug ml of azaguanine in complete growth medium. About 95% of the azaguanine resistant clones were sensitive... 

Table 1. Influence of inhibition of poly(ADP-ribose) polymerase on MNNG X-ray and UV-induced 8-azaguanine resistant mutant in V79 cells...

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