Effect of 5-Azacytidine on Malignant Transformation of a Mutant Derived from the Mouse BALB/c 3T3 Cell Line Resistant to Transformation by Chemical Carcinogens

BALB/c 3T3 and its derivative MO-5, isolated as a monensin-resistant clone, showed a very low rate of spontaneous malignant transformation. Treatment of BALB/c 3T3 cells with benzo( a )pyrene, 4-nitroquinoline- N -oxide, N -methyl- N ′-nitro- N -nitrosoguanidine, N -methyl- N -nitrosourea, or UV light irradiation significantly enhanced the rate of transformation, whereas the treatment of MO-5 cells with these carcinogens had only a slight if any effect. Exposure of MO-5 as well as BALB/c 3T3 to 5 or 10 µm 5-azacytidine for 3 to 7 days significantly increased the number of transformation foci. The Luria-Delbruck fluctuation test showed that spontaneous mutation frequency (mutants/cell/generation) was 1.2 × 10-6 for BALB/c 3T3 and 7.1 × 10-7 for MO-5, respectively, when appearance of cadmium-resistant clones was tested. N -Methyl- N ′-nitro- N -nitrosoguanidine enhanced induced mutation frequency of ouabain-resistant and cadmium-resistant mutants of BALB/3T3 but it only slightly enhanced that of MO-5. Methylation status of DNA of MO-5 was compared with that of BALB/c 3T3 by comparing the cleavage patterns generated by the isoschizomeric restriction enzymes Hpa II and Msp I. DNA of MO-5 was found to be more methylated than that of BALB/c 3T3 in the vicinity of c- myc as well as the metallothionein-I gene. Aberrant DNA methylation in MO-5 and the cellular sensitivity to transformation by chemical carcinogens or 5-azacytidine are discussed.