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.Although environmental agents have been shown to play an important role in the initiation of most human cancers, little is known about the molecular mechanisms underlying their action. Tumorigenesis is accompanied by a variety of molecular processes including DNA rearrangements (18, 21), oncogene activation (2) and amplification (5,24,25), and the appearance of double-minute chromosomes and homogeneously staining regions containing amplified DNA sequences (7,10,16).In recent years we studied the effect of chemical and physical carcinogens on one of these cancer-related phenomena, namely, gene amplification, in an attempt to establish its possible role in the initiation events of carcinogenesis. To study gene amplification immediately after exposure to the carcinogens rather than after the long process of establishing cell lines containing amplified genes, we have constructed an experimental model system consisting of simian virus 40 (SV40)-transformed Chinese hamster cells (C060) (12). In this system SV40 amplification can be monitored by molecular hybridization. Exposure of these cells to a variety of chemical and physical carcinogens has been shown to result in the amplification of SV40 DNA sequences. This amplification requires an active origin of replication and a functional T antigen. The amplification phenomenon is transient, reaching a maximal level on the third and fourth day after treatment with carcinogen, and disappears within a few more days (12,14,15). Exposure to carcinogens is followed by a prolonged S phase. Treatment of a synchronized population of cells obtained by isoleucine starvation results in increased SV40 amplification (Y. Berko and S. Lavi, manuscript in preparation).Further studies in our laboratory revealed transient amplification of additional DNA sequences including the dhfr and rasH genes in response to carc...

Small polydispersed circular DNA (spcDNA) in human cells: association with genomic instability

“…Increased quantities of spcDNA were also detected following treatments of cultured mouse cells with the drug cycloheximide or with agents arresting DNA replication, such as hydroxyurea (HU) or 7,1-dimethylbenzanthracene (DMBA) plus naladixic acid (Sunnerhagen et al, 1986(Sunnerhagen et al, , 1989. Carcinogens were also found to enhance SV40 ampli®cation in CO60 cells (Lavi, 1982;Lavi and Etkin, 1981) involving the formation of circular molecules (Cohen and Lavi, 1996), and were reported to induce cellular gene ampli®cation in dierent cell lines (Kleinberger et al, 1986;Schimke, 1988). spcDNA amounts are enhanced in several conditions associated with genomic instability, such as angiofibromas derived from Tuberous Sclerosis patients (Neidlinger et al, 1988) and cells from Fanconi's Anemia patients (Motejlek et al, 1993).…”

“…This happens in tumor-prone genetic diseases, such as Fanconi's Anemia (FA), Xeroderma Pigmentosum (XP) and Ataxia Talangiectasia (AT), where defective DNA repair leads to genomic instability, preceding cancer (for a review see Hartwell, 1992). Genomic instability was also shown to be induced by cancer initiating agents, such as chemical and physical carcinogens (Kleinberger et al, 1986;Tlsty, 1982).…”

Mithramycin inhibits SP1 binding and selectively inhibits transcriptional activity of the dihydrofolate reductase gene in vitro and in vivo.

“…DHFR gene amplification has been demonstrated in a number of methotrexate-resistant human (37-39) and animal (25,33,(40)(41)(42)(43) cell lines, and documented clinically in a number of cases of methotrexate resistant neoplasms (44)(45)(46). The methotrexate resistant cells used in this work have an 25-fold increase in DHFR gene copy number, mRNA, and enzyme activity, but a disproportionately high (l0,OOOX) increase in methotrexate resistance (20).…”

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.…”