Inhibition of N‐methyl‐N′'‐nitro‐n‐nitrosoguanidine‐induced methotrexate and adriamycin resistance in CHO cells by adeno‐associated virus type 2

Abstract

We studied the effects of helper-dependent parvovirus AAV [adeno-associated virus] type 2 on carcinogen-inducible resistance to methotrexate (MTX) and adriamycin (ADR) in Chinese hamster ovary cells. Both types of drug resistance were monitored by determination of the number of drug-resistant colonies normalized for the respective value of plating efficiency under non-selective conditions. Treatment of cells with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) drastically enhanced the frequency of resistance to MTX and ADR. By contrast, infection of cells with AAV-2 prior to treatment with MNNG markedly inhibited carcinogen-induced drug resistance. Infection by AAV alone did not exert any effect. Analysis of the dihydrofolate reductase (dhfr) gene copy numbers of individual MTX-resistant clones derived from MNNG-treated and non-treated cultures revealed similar frequencies (60-80%) and amplitudes of dhfr gene amplification (2- to 8-fold) irrespective of prior AAV treatment. Hence, carcinogen-induced enhancement of MTX-resistance could reflect an increase in the frequency of dhfr gene amplification among the survivors of MNNG treatment. On the other hand, inhibition of carcinogen-inducible drug resistance by AAV suggests an interference of the virus with cellular responses to genotoxic stress, thus leading to enhanced cell killing under altered growth conditions. Possible mechanisms responsible for the inhibitory effect of AAV and its relevance in relation to tumor chemotherapy are discussed.