We propose that a fundamental problem in the faithful replication of complex chromosomes of higher eukaryotes is the proper control of both the number and timing of the multiple initiations of replication on single chromosomes. When replication patterns are disrupted by any of a variety of agents, overreplication of DNA can occur. We propose a model that accounts for the generation of a wide variety of chromosomal aberrations-rearrangements, resulting from the various ways in which the overreplicated strands can undergo recombination. We also discuss certain implications of the generation of chromosomal alterations in higher eukaryotes as they may relate to cancer chemotherapy, cancer progression, aging, and rapid speciation-evolution.Previous studies in our laboratory have examined the process of gene amplification in cultured mammalian cells (for reviews, see refs. 1 and 2). The frequency of a spontaneous doubling of the dihydrofolate reductase (DHFR) gene is 1 x io-, per cell generation (3), and this frequency can be increased 10-fold or more by pretreatment of cells with agents such as hydroxyurea (4), UV light, and carcinogens (5). Mariani and Schimke (6) provided evidence that when DNA synthesis was inhibited during hour 2 of S phase in synchronized Chinese hamster ovary (CHO) cells, DNA synthesized before the hydroxyurea block was rereplicated once the hydroxyurea was removed. This overreplication resulted in an increase in the number of DHFR genes and led to enhanced resistance to methotrexate (MTX). This overreplication involves not only the DHFR gene, which is replicated early in S phase, but also a large proportion of the DNA replicated prior to the inhibition of DNA synthesis.More recently Hill and Schimke (7) have extended these studies to show that treatment of cells with hydroxyurea results in the generation of a wide variety of chromosomal aberrations-rearrangements observed in the first M phase following inhibition of DNA synthesis. The aberrations include normal chromosomes with extrachromosomal DNA, increased frequencies of sister chromatid exchange, polyploidization, breakage-bridge fusion chromosomes, and gapped-fragmented chromosomes. Most significant in their results is the observation that the cells in which the chromosomal alterations are observed are derived from that subset of the treated cells that contains more than the G2 content of DNA as studied with DNA fluorochromes and flow cytometric techniques.We observed additionally (S.W.S., A.B.H., and R.T.S., unpublished data) that the increased DNA content of cells previously treated with hydroxyurea (or aphidicolin) does not result from fusion of cells or uptake of DNA from killed cells. The finding that the chromosomal alterations occur only within the subset of cells with additional DNA leads us to propose that the chromosomal alterations are the consequence of recombination events involving the strands of overreplicated DNA, and not that the additional DNA occurs after the generation of the chromosomal aberrations. Thus, we p...
Melatonin has been shown to have a direct inhibitory action on the proliferation of estrogen-responsive MCF-7 human breast cancer cells in culture. In the present study, we examined by flow cytometry whether this inhibitory effect might be exerted on the G1 phase of the cell cycle, thus causing a transition delay into the S phase. In order to further verify this hypothesis we tested the ability of estradiol to "rescue" MCF-7 cells from melatonin inhibition, and the potential of this indoleamine to block the ability of estradiol to rescue the cells from tamoxifen inhibition. Following five days of incubation, melatonin (10(-9)M) increased the fraction of cells in G1 of the cell cycle while simultaneously causing a 50% reduction in the proportion of cells in S phase. The antiproliferative effect of melatonin (10(-5)M) was prevented by the simultaneous treatment of the cells with estradiol (10(-8)M) in clonogenic soft agar culture, or reversed by the addition of estradiol to cells previously incubated with and inhibited by melatonin (10(-9)M) in monolayer culture. Additionally, melatonin blocked the estrogen-rescue of tamoxifen-inhibited cells in both types of culture systems. These results support the hypothesis that the antiproliferative effect of melatonin, like tamoxifen, is cell cycle specific by causing a G1-S transition delay. These results also indicate an important interaction of melatonin with estrogen-mediated mechanisms of MCF-7 cell proliferation.
We examined the role that blockage of cells in the cell cycle may play in the stimulation of gene amplification and enhancement of drug resistance. We found that several different inhibitors of DNA synthesis, which were each able to block cells at the G1-S-phase boundary, induced an enhanced cycloheximide-sensitive synthesis of an early S-phase cell cycle-regulated enzyme, dihydrofolate reductase, and of other proteins as well. This response was specific, in that blockage at the G2 phase did not result in overproduction of the enzyme. When the cells were released from drug inhibition, DNA synthesis resumed, resulting in a cycloheximide-sensitive elevation in DNA content per cell. We speculate that the excess DNA synthesis (which could contribute to events detectable later as gene amplification) is a consequence of the accumulation of S-phase-specific proteins in the affected cells, which may then secondarily influence the pattern of DNA replication.
Summary Gene amplification is a mechanism whereby cultured animal cells and human tumours become resistant to cancer chemotherapeutic agents. This review of studies from the authors' laboratory describes properties of the acquisition of resistance to methotrexate in cultured mammalian cells by virtue of amplification of the dihydrofolate reductase gene. These properties result in a heterogeneous cell population with respect to many cell properties, including the number and stability of the amplified genes. Gene amplification results from overreplication of DNA in a single cell cycle as a result of inhibition of DNA synthesis. The cells surviving such overreplication constitute a heterogeneous population with multiple chromosomal changes, including partial or complete endoreduplication of chromosomes, as well as a variety of chromosomal rearrangements. A similar phenomenon may underlie the generation of aneuploidy in tumours, their malignant progression, and the generation of heterogeneity in the tumour cell population.
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