The Dual Effect of Mimosine on DNA Replication

Kalejta, Robert F.; Hamlin, Joyce L.

doi:10.1006/excr.1996.3444

Cited by 52 publications

(29 citation statements)

References 47 publications

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“…CHO cells can efficiently be arrested in the G1/S phase by mimosine, a drug that reversibly inhibits both replication initiation and strand elongation. 21 After removing the drug, cells progress synchronically into the S and G2/M phase. 22 As seen in Figure 3a (right panel), cells kept in the G1/S phase by continuous exposure to mimosine before and after transfection with ssODN displayed no sequence correction.…”

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Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides

Olsen¹,

Randøl²,

Krauß³

2005

Gene Ther

106

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Oligonucleotide-based sequence alteration in living cells is a substantial methodological challenge in gene therapy. Here, we demonstrate that using corrective single-stranded oligonucleotides (ssODN), high and reproducible sequence correction rates can be obtained. CHO cell lines with chromosomally integrated multiple copy EGFP reporter genes routinely show rates of 4.5% targeted sequence correction after transfection with ssODN. We demonstrate that the cell cycle influences the rates of targeted sequence correction in vivo, with a peak in the early S phase during ssODN exposure. After cell division, the altered genomic sequence is predominantly passed to one daughter cell, indicating that targeted sequence alteration occurs after the replication fork has passed over the targeted site. Although high initial correction rates can be obtained by this method, we show that a majority of the corrected cells arrest in the G2/M cell cycle phase, although 1-2% of the corrected cells form viable colonies. The G2/M arrest observed after targeted sequence correction can be partially released by caffeine, pentoxifylline or Gö 6976 exposure. Despite substantial remaining challenges, targeted sequence alteration based on ssODN increasingly promises to become a powerful tool for functional gene alterations. Gene Therapy (2005) Various strategies have been developed to achieve therapeutic targeted gene repair, including small fragment homologous sequence 1,2 ssODN, 3-6 triple helixforming oligonucleotides containing a reactive group, 7-9 bifunctional oligonucleotides based on a triple helixforming DNA recognition domain 10,11 and branched oligonucleotides. 12 Although a general proof of principle for oligonucleotide-based sequence alteration appears to be established, the paradigm for oligonucleotide-based genome alteration remain largely unknown. 6 The central problem in studying this phenomenon are low rates of in vivo sequence correction rates in cell culture. Generally, observed sequence alteration rates on in vivo chromosomal templates in selected cell lines are in the range of 0.1%, 6 while correction rates in untransformed primary cells are substantially lower. [13][14][15] In the absence of a suitable selection system, such low rates allow only limited functional analysis, although Yoon and co-workers have recently suggested a selection system based on simultaneous targeting of two loci. 16 A further problem is the variety of model systems and assays that were used to measure targeted sequence correction which lead to confusing and often conflicting results. 6 Despite these obstacles, several consistent observations have been made: targeted sequence correction depends centrally on transcription, and ssODN that are complementary to the nontranscribed strand show in most cases substantially higher correction rates than ssODN complementary to the transcribed strand in both episomal and chromosomal assays. 17,18 The involvement of transcription-coupled repair has therefore been suggested. Furthermore, the absence of MSH2, ...

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“…Cells were arrested at the G1/S border by the procedure described. 21 Briefly, the cells were grown for 48 h in media containing 0.2% FBS, then incubated for 24 h in growth media containing 5% FBS and 100 mM mimosine (Sigma). The cells were transfected (1 h) in media containing mimosine.…”

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confidence: 99%

Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides

Olsen¹,

Randøl²,

Krauß³

2005

Gene Ther

106

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“…Other studies demonstrate that mimosine disrupts the initiation phase of DNA replication at the origins of replication in Chinese hamster ovary cells. Mimosine is novel in this regard; it prevents formation of replication intermediates (4,10). Mimosine also affects gene expression at the level of transcription and translation.…”

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Mimosine Attenuates Serine Hydroxymethyltransferase Transcription by Chelating Zinc

Perry

Sastry

Nasrallah

et al. 2005

Journal of Biological Chemistry

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L-Mimosine is a naturally occurring plant amino acid and iron chelator that arrests the cell cycle in the late G 1 phase, although its mechanism of action is not known. Some studies indicate that mimosine prevents the initiation of DNA replication, whereas other studies indicate that mimosine disrupts elongation of the replication fork by impairing deoxyribonucleotide synthesis by inhibiting the activity of the iron-dependent enzyme ribonucleotide reductase and the transcription of the cytoplasmic serine hydroxymethyltransferase gene (SHMT1). In this study, the mechanism for mimosineinduced inhibition of SHMT1 transcription was elucidated. A mimosine-responsive transcriptional element was localized within the first 50 base pairs of the human SHMT1 promoter by deletion analyses and gel mobility shift assays. The 50-base-pair sequence contains a consensus zinc-sensing metal regulatory element (MRE) at position ؊44 to ؊38, and mutation of the MRE attenuated mimosine-induced transcription repression. Mimosine treatment eliminated MRE-and Sp1-binding activity in nuclear extracts from MCF-7 cells but not in nuclear extracts from a mimosine-resistant cell line, MCF-7/2a. MCF-7 cells cultured in zinc-depleted medium for more than 16 days were viable and lacked cytoplasmic serine hydroxymethyltransferase protein, confirming that mimosine inhibits SHMT1 transcription by chelating zinc. The disruption of DNA-protein interactions by zinc chelation provides a general mechanism for the inhibitory effects of mimosine on nuclear processes, including replication and transcription. Furthermore, this study establishes that SHMT1 is a zinc-inducible gene, which provides the first mechanism for the regulation of folate-mediated one-carbon metabolism by zinc.Mimosine is an amino acid found in plants of the Mimosa and Leucaena genera and a tyrosine analog (1). It is a cell-specific inhibitor of DNA replication in vivo; sensitive cell lines include human MCF-7 (2), HL60 (3) and Chinese hamster ovary (4), whereas human neuroblastoma (2), K562 (3), and Xenopus and mouse embryonic cells are resistant (5, 6). Mimosine is an effective iron chelator and a structural analog of deferiprone, a compound that is used clinically to lower systemic iron in patients who suffer from iron overload (7, 8) (Fig. 1).Mimosine arrests cell cycle progression in late G 1 and although it elicits a plethora of biological effects, its mechanism of action has not been established. Mimosine has been proposed to arrest DNA synthesis at the replication fork by repressing deoxyribonucleotide synthesis (9 -12). Mimosine and deferoxamine inhibit the activity of the iron-dependent enzyme ribonucleotide reductase in vitro by chelating iron (9, 11, 12) and deplete cellular deoxyribonucleotide pools in some but not all studies (2, 12). Furthermore, mimosine and deferoxamine have been shown to inhibit the transcription of the cytoplasmic serine hydroxymethyltransferase gene (SHMT1) 1 in MCF-7 cells but not in mimosine-resistant SH-SY5Y neuroblastoma (2). The cytoplasmic ser...

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“…1), is a toxin that chelates iron and inhibits mammalian DNA replication. Mimosine is known to block DNA replication in breast cancer cells and Chinese hamster ovary cells at the initiation phase, although, the precise mechanism by which mimosine alters DNA replication remains unclear (8). Recently, Alpan and Pardee (11) proposed a mechanism to account for the effects of mimosine on DNA replication.…”

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“…However, other data do not support this mechanism. Inhibition of RNR would not be expected either to inhibit the initiation phase of DNA replication (8) or to specifically lower purine deoxyribonucleotide pools. Furthermore, the effect of mimosine on DNA replication is cell-specific.…”

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Mimosine Is a Cell-specific Antagonist of Folate Metabolism

Oppenheim

Nasrallah

Mastri

et al. 2000

Journal of Biological Chemistry

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Iron deficiency and iron chelators are known to alter folate metabolism in mammals, but the underlying biochemical mechanisms have not been established. Although many studies have demonstrated that the iron chelators mimosine and deferoxamine inhibit DNA replication in mammalian cells, their mechanism of action remains controversial. The effects of mimosine on folate metabolism were investigated in human MCF-7 cells and SH-SY5Y neuroblastoma. Our findings indicate that mimosine is a folate antagonist and that its effects are cell-specific. MCF-7 cells cultured in the presence of 350 M mimosine were growth-arrested, whereas mimosine had no effect on SH-SY5Y cell proliferation. Mimosine altered the distribution of folate cofactor forms in MCF-7 cells, indicating that mimosine targets folate metabolism. However, mimosine does not influence folate metabolism in SH-SY5Y neuroblastoma. The effect of mimosine on folate metabolism is associated with decreased cytoplasmic serine hydroxymethyltransferase (cSHMT) expression in MCF-7 cells but not in SH-SY5Y cells. MCF-7 cells exposed to mimosine for 24 h have a 95% reduction in cSHMT protein, and cSHMT promoter activity is reduced over 95%. Transcription of the cSHMT gene is also inhibited by deferoxamine in MCF-7 cells, indicating that mimosine inhibits cSHMT transcription by chelating iron. Analyses of mimosine-resistant MCF-7 cell lines demonstrate that although the effect of mimosine on cell cycle is independent of its effects on cSHMT expression, it inhibits both processes through a common regulatory mechanism.There are several well characterized cellular responses that are triggered following decreases in the regulatory, non-ferritin-bound iron pool. Many of these responses are mediated through the iron regulatory protein, which can bind with specificity to certain mRNA species and regulate translation (1). The concentration of cellular regulatory iron is decreased by iron deficiency or by elevated expression of heavy chain ferritin, a protein that sequesters intracellular iron (2, 3). Sequestration of intracellular iron by chelators, including DFO, 1 has been commonly used to trigger physiological changes associated with iron deficiency. The influence of iron deficiencies, both induced and naturally occurring, on folate metabolism has been well documented in cell culture models, animal models, and humans. Iron deficiency can result in morphological alterations in granulocytes similar to folate deficiency (4), and iron deficiency has been demonstrated to impair folate utilization in some but not all tissues (5). In addition, maternal iron deficiency decreases secretion of folate into milk (6, 7) without decreasing maternal serum or red blood cell folate levels in rats. However, the biochemical mechanisms underlying the influence of iron deficiency on folate metabolism have not been established (6).Mimosine, a plant amino acid and tyrosine analog ( Fig. 1), is a toxin that chelates iron and inhibits mammalian DNA replication. Mimosine is known to block DNA replication...

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The Dual Effect of Mimosine on DNA Replication

Cited by 52 publications

References 47 publications

Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides

Implications of cell cycle progression on functional sequence correction by short single-stranded DNA oligonucleotides

Mimosine Attenuates Serine Hydroxymethyltransferase Transcription by Chelating Zinc

Mimosine Is a Cell-specific Antagonist of Folate Metabolism

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