Thymidylate stress induces homologous recombination activity in mammalian cells

doi:10.1016/0027-5107(91)90124-7

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volume 246, Issue 1, January 1991, Pages 215-220

https://doi.org/10.1016/0027-5107(91)90124-7 Get rights and content

Abstract

We studied whether homologous recombination activity in mammalian cells could be induced by thymidylate stress (thymidylate deprivation). In vitro recombination activity in cell extracts was measured with pSV2neo-derived plasmids. When prior to the preparation of extracts, mouse FM3A cells were grown in 5-fluorodeoxyuridine (FdUrd), an inducer of thymidylate stress, the homologous recombination activity was significantly induced, as judged from an increase in the number of neomycin-resistant bacterial colonies. Maximum induction was observed in cells treated with 1 μM FUdR for 16h. However, 3–8 h of treatment of FM3A cells with the drug followed by an additional 8–16-h incubation in its absence was sufficient to induce the recombination activity while slightly reducing their growth rates. These results indicate that thymidylate stress induces homologous recombination activity in mammalian cells as observed in Escherichia coli and in yeast.

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Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells
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Genetic and biochemical consequences of thymidylate stress
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Ability of structurally related polycyclic aromatic carcinogens to induce homologous recombination between duplicated chromosomal sequences in mouse L cells
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Isolation and characterization of recombination-deficient mutants of Escherichia coli K12

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Measurement of the stability of the repressor of alkaline phosphatase synthesis in Escherichia coli

R.H. Haynes

Molecular mechanisms in genetic stability and change

Cited by (11)

DNA damage and homologous recombination signaling induced by thymidylate deprivation
2008, Biochemical Pharmacology
Citation Excerpt :
Unresolved BER strand break intermediates appear to be processed by HR [9]. Thymidylate deprivation in S. cerevisiae has been shown to induce recombination [10], and there is evidence in murine cells that thymidylate deprivation can induce events suggestive of recombination [11,12]. Collectively, the observations suggest that HR is likely involved in the response to thymidylate deprivation in mammalian cells.
DNA damage is accepted as a consequence of thymidylate deprivation induced by chemotherapeutic inhibitors of thymidylate synthase (TS), but the types of damage and signaling responses remain incompletely understood. Thymidylate deprivation increases dUTP and uracil in DNA, which is removed by base excision repair (BER). Because BER requires a synthesis step, strand break intermediates presumably accumulate. Thymidylate deprivation also induces cell cycle arrest during replication. Homologous recombination (HR) is a means of repairing persistent BER intermediates and collapsed replication forks. There are also intimate links between HR and S-phase checkpoint pathways. In this study, the goals were to determine the involvement of HR-associated proteins and DNA damage signaling responses to thymidylate deprivation. When RAD51, which is a central component of HR, was depleted by siRNA cells were sensitized to raltitrexed (RTX), which specifically inhibits TS. To our knowledge, this is the first demonstration in mammalian cells that depletion of RAD51 causes sensitivity to thymidylate deprivation. Activation of DNA damage signaling responses was examined following treatment with RTX. Phosphorylation of replication protein A (RPA2 subunit) and formation of damage-induced foci were strikingly evident following IC₅₀ doses of RTX. Induction was much more striking following RTX treatment than with hydroxyurea, which is commonly used to inhibit replication. RTX treatment also induced foci of RAD51, γ-H2AX, phospho-Chk1, and phospho-NBS1, although the extent of co-localization with RPA2 foci varied. Collectively, the results suggest that HR and S-phase checkpoint signaling processes are invoked by thymidylate deprivation and influence cellular resistance to thymidylate deprivation.
Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase
2008, DNA Repair
Thymidylate deprivation brings about “thymineless death” in prokaryotes and eukaryotes. Although the precise mechanism for thymineless death has remained elusive, inhibition of the enzyme thymidylate synthase (TS), which catalyzes the de novo synthesis of TMP, has served for many years as a basis for chemotherapeutic strategies. Numerous studies have identified a variety of cellular responses to thymidylate deprivation, including disruption of DNA replication and induction of DNA breaks. Since stalled or collapsed replication forks and strand breaks are generally viewed as being recombinogenic, it is not surprising that a link has been demonstrated between recombination induction and thymidylate deprivation in bacteria and lower eukaryotes. A similar connection between recombination and TS inhibition has been suggested by studies done in mammalian cells, but the relationship between recombination and TS inhibition in mammalian cells had not been demonstrated rigorously. To gain insight into the mechanism of thymineless death in mammalian cells, in this work we undertook a direct investigation of recombination in human cells treated with raltitrexed (RTX), a folate analog that is a specific inhibitor of TS. Using a model system to study intrachromosomal homologous recombination in cultured fibroblasts, we provide definitive evidence that treatment with RTX can stimulate accurate recombination events in human cells. Gene conversions not associated with crossovers were specifically enhanced several-fold by RTX. Additional experiments demonstrated that recombination events provoked by a double-strand break (DSB) were not impacted by treatment with RTX, nor was error-prone DSB repair via nonhomologous end-joining. Our work provides evidence that thymineless death in human cells is not mediated by corruption of DSB repair processes and suggests that an increase in chromosomal recombination may be an important element of cellular responses leading to thymineless death.
Determination of apoptosis, uracil incorporation, DNA strand breaks, and sister chromatid exchanges under conditions of thymidylate deprivation in a model of BER deficiency
2005, Biochemical Pharmacology
Citation Excerpt :
However, the oligonucleotide-based assay only provides a measure of overall uracil DNA glycosylase activity, and does not distinguish between the different uracil DNA glycosylases (e.g., UDG, SMUG), nor between the nuclear and mitochondrial forms of UDG. Thymidylate deprivation induced by FdUrd has been reported to induce genetic recombination [40,41]. Although substantial strand break formation was not detected at earlier time points or lower doses of RTX (above), we sought to determine whether RTX might transiently induce strand break events that went undetected by Comet assay or PFGE.
Thymidylate synthase (TS) is an important target of several chemotherapeutic agents. During TS inhibition, dTTP levels decrease with a subsequent increase in dUTP. Uracil incorporated into the genome is removed by base excision repair (BER). BER has been hypothesized to play a role in the response to thymidylate deprivation, despite a lack of direct evidence. We previously found that β-pol null murine fibroblasts were ∼six-fold more resistant than wild-type cells to raltitrexed, a folate-based inhibitor specific for TS. In this study, a number of endpoints were determined to understand the influence of BER and β-pol during raltitrexed treatment. Raltitrexed induced apoptosis in wild-type cells to a greater extent than in β-pol null cells. A PARP inhibitor decreased the sensitivity to raltitrexed, although the extent was not different between wild-type and β-pol null cells. No evidence was seen for extensive strand break formation that preceded apoptosis, although raltitrexed induced more sister chromatid exchanges in wild-type cells. Increased levels of uracil in DNA were detected following treatment in wild-type and β-pol null cells. However, uracil levels were only ∼two-fold higher in DNA from treated cells compared to untreated. Uracil DNA glycosylase activity was slightly higher in β-pol null cells, although not sufficiently different to explain the difference in sensitivity to raltitrexed. Taken together, the data suggest that the sensitivity of the wild-type cells to raltitrexed is not associated with activation of PARP-1 dependent BER, extensive uracil incorporation into DNA and persistent strand breaks, but rather with changes suggestive of DNA recombination.
Uracil misincorporation, DNA strand breaks, and gene amplification are associated with tumorigenic cell transformation in folate deficient/repleted Chinese hamster ovary cells
1999, Cancer Letters
Clinical and experimental evidence has linked nutritional folic acid status to both anti- and procarcinogenic activity. Folate supplementation of normal cells appears to have a protective effect; however, folate supplementation of initiated cells may promote neoplastic progression. Given these considerations, the present series of experiments examines alterations in DNA metabolism and cumulative DNA lesions using an in vitro model of folate deprivation and repletion. DNA repair-deficient CHO-UV5 cells were cultured in Ham's F-12 medium or in custom-prepared Ham's F-12 medium lacking in folic acid, thymidine and hypoxanthine for a period of 18 days without cell passage. The results indicated that progressive folate and nucleotide depletion leads to a significant increase in the ratio of dUTP/dTTP and to the misincorporation of uracil into DNA. These alterations were accompanied by growth inhibition, DNA strand breaks, abasic sites and phenotypic abnormalities. After 14 days in culture, there was significant increase in gene amplification potential in the chronically folate-deficient cells, but no significant increase in anchorage-independent growth or in neoplastic transformation. Acute folate repletion of the deficient cells was used as a proliferative stimulus under conditions of dNTP pool imbalance and multiple lesions in DNA. A further increase in gene amplification was accompanied by anchorage-independent growth and neoplastic cell transformation as evidenced by aggressive tumor growth in Balb/c nu/nu mice. Using a sensitive in vitro model system, these results emphasize the essentiality of folic acid for de novo nucleotide synthesis and the integrity of the DNA. However, the in vivo relevance, especially in terms of tumorigenic potential, is not clear.
A plasmid system to monitor gene conversion and reciprocal recombination in vitro
1993, Mutation Research/Environmental Mutagenesis and Related Subjects
A plasmid system allowing for the detection of recombinagenic activitues in cell-free extracts is described. Two truncated alleles of the bacterial neomycin resistance gene (neo), differing from each other at a polymorphic restriction site, were constructed. Recombinations involving both alleles mediated by Drosophila embryo nuclear protein extracts or Drosophila larva whole cell protein extracts were selected by their ability to confer kanamycin resistance to E. coli. Restriction analysis of plasmids recovered from E. coli transformants allowed the monitoring of the two molecular mechanisms which can lead to functional neo genes, gene conversion and reciprocal recombination.
A dose dependent increase in the recombination frequency with increasing amounts of cell extract was observed. Recombination was further increased by linearizing one of the two substrate plasmids. The Drosophila cell extracts catalyzed recombination in vitro since after incubation a recombination product could be identified by polymerase chain reaction (PCR) technology. The recombination was absolutely dependent on the presence of an active cell extract, since no diagnostic PCR product was detected in a reaction where extract was omitted. Analysis of a representative number of recombinant plasmids by restriction analysis revealed that in the absence of an exogenous recombinational system less than 2% of kanamycin resistant recombinant plasmids occurred by gene conversion upon transformation into E. coli. In contrast, recombinants exhibiting restriction patterns diagnostic for gene conversion were observed at frequencies between 5.1% and 9.8% after incubation with Drosophila larva cell extracts. These results strongly argued that gene conversion is a prominent mechanism of recombination in Drosophila mitotic cells.
Mutagen-induced recombination in mammalian cells in vitro
1992, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
It is now clear from in vitro studies that mutagens induce recombination in the cell, both homologous and nonhomologous exchanges. The recombination events induced are extrachromosomal events, exchanges between extrachromosomal DNA and chromosomes, and inter- as well as intrachromosomal exchanges. However, not all types of DNA damage can induce recombination. The mechanisms involved in the induction process are not known but may involve activation of DNA repair systems. In addition, stimulation of mRNA transcription by mutagens, different recombination pathways and how the assay system is constructed may affect the frequency and characteristics of the observed recombination events.

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Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Abstract

References (27)

Accumulation of DNA strand breaks during thymineless death in thymidylate synthase-negative mutants of mouse FM3A cells

J. Biol. Chem.

A specialized form of chromosomal DNA degradation induced by thymidylate stress in mouse FM3A cells

Mutation Res.

Thymidineless death and mutation induction in cultured mouse FM3A cell mutants deficient in thymidylate synthase

Mutation Res.

DNA repair and the genetic effects of thymidylate stress in yeast

Mutation Res.

Deoxyribonucleoside triphosphate imbalance: 5-fluorodeoxyuridine-induced DNA double strand breaks in mouse FM3A cells and the mechanism of cell death

J. Biol. Chem.

Selection of mammalian thymidine auxotropic cell mutants defective in thymidylate synthase by their reduced sensitivity to methotrexate

Somat. Cell Genet.

Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells

Mol. Cell. Biol.

Genetic and biochemical consequences of thymidylate stress

Can. J. Biochem.

Ability of structurally related polycyclic aromatic carcinogens to induce homologous recombination between duplicated chromosomal sequences in mouse L cells

Mutation Res.

Isolation and characterization of recombination-deficient mutants of Escherichia coli K12

Studies on unbalanced growth in Escherichia coli

Measurement of the stability of the repressor of alkaline phosphatase synthesis in Escherichia coli

Molecular mechanisms in genetic stability and change

Cited by (11)

DNA damage and homologous recombination signaling induced by thymidylate deprivation

Induction of intrachromosomal homologous recombination in human cells by raltitrexed, an inhibitor of thymidylate synthase

Determination of apoptosis, uracil incorporation, DNA strand breaks, and sister chromatid exchanges under conditions of thymidylate deprivation in a model of BER deficiency

Uracil misincorporation, DNA strand breaks, and gene amplification are associated with tumorigenic cell transformation in folate deficient/repleted Chinese hamster ovary cells

A plasmid system to monitor gene conversion and reciprocal recombination in vitro

Mutagen-induced recombination in mammalian cells in vitro