Carcinogen-Mediated Methotrexate Resistance and Dihydrofolate Reductase Amplification in Chinese Hamster Cells

Abstract

We have investigated different parameters characterizing carcinogen-mediated enhancement of methotrexate resistance in Chinese hamster ovary (CHO) cells and in simian virus 40-transformed Chinese hamster embryo (C060) cells. We show that this enhancement reflects dihydrofolate reductase (dhfr) gene amplification. The carcinogens used in this work are alkylating agents and UV irradiation. Both types of carcinogens induce a transient enhancement of methotrexate resistance which increases gradually from the time of treatment to 72 to 96 h later and decreases thereafter. Increasing doses of carcinogens decrease cell survival and increase the enhancement of methotrexate resistance. Enhancement was observed when cells were treated at different stages in the cell cycle, and it was maximal when cells were treated during the early S phase. These studies of carcinogen-mediated dhfr gene amplification coupled with our earlier studies on viral DNA amplification in simian virus 40-transformed cells demonstrate that the same parameters characterize the amplification of both genes. Possible cellular mechanisms responsible for the carcinogen-mediated gene amplification phenomenon are discussed.

Binding of a Sequence-Specific Single-Stranded DNA-Binding Factor to the Simian Virus 40 Core Origin Inverted Repeat Domain is Cell Cycle Regulated

“…In synchronized SV40 permissive cells, only cell extracts prepared from cells after Gl-to-S-phase (G,/S-phase) transition are capable of supporting SV40 DNA synthesis in vitro (37). Similarly, amplification of the dihydrofolate reductase gene induced by methotrexate or amplification of SV40 in response to DNA damage occurs maximally when the cells are treated in early S-phase (21). This cell cycle dependence occurs despite the fact that many of the key DNA replication proteins appear to be constitutively expressed throughout the proliferative cell cycle (10,30,32,37,55,59).…”

Hairpin Structures Are the Primary Amplification Products: a Novel Mechanism for Generation of Inverted Repeats during Gene Amplification

“…The carcinogen treatment of the semipermissive Chinese hamster cells is essential for SV40 amplification both in vivo and in vitro, since cellular factors responsible for the initiation of SV40 replication are activated in the treated cells (1,4,28). Previously, we have shown that in the treated cells dihydrofolate reductase amplification is enhanced and shares many characteristics with the SV40 amplification, thus suggesting that the same cellular factors control the SV40 amplification and the amplification of cellular genes (24,30).…”

“…The replication is bidirectional, and the amplified DNA is heterogeneous in size and enriched for sequences spanning the region around the SV40 origin of replication (29). Carcinogenenhanced gene amplification was demonstrated for several cellular genes (24,52), and it is similar to SV40 amplification in numerous characteristics (24,30).…”

Carcinogen-Induced trans Activation of Gene Expression

“…Other processes, such as chromosomal rearrangements (32), gene amplification (2), and changes in gene expression (39), are also utilized in normal cell regulation and differentiation (6,33,34,37); however, the divergence of these processes from their natural patterns of occurrence might be related to the carcinogenic process. Carcinogens cause phenomena which accompany malignancy (1,3,5,17,19,22,36). We reported previously that carcinogens induce gene amplification and enhanced gene expression of simian virus 40 (SV40) and dihydrofolate reductase (17, 19, 20, 20a).…”

“…Gels were dried and fluorographed. SV40 amplification was measured by slot blot hybridization on day 3 after treatment, which was the time of maximal carcinogen-induced amplification (a and b) (17,19). C, Control cells; T, treated cells; Aph., aphidicolin.…”