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Treatment of mammalian cells with mimosine generates DNA breaks
Mutation Research 459 Ž2000. 299–306 www.elsevier.comrlocaterdnarepair Community address: www.elsevier.comrlocatermutres
Treatment of mammalian cells with mimosine generates DNA breaks Ivailo Mikhailov, George Russev, Boyka Anachkova) Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. BoncheÕ Street, Bl. 21, 1113 Sofia, Bulgaria Received 6 September 1999; received in revised form 4 February 2000; accepted 4 February 2000
Abstract Exponentially growing mouse erythroleukemia ŽMEL. cells and quiescent human peripheral blood lymphocytes ŽPBL. were treated with different concentrations of the nonprotein amino acid mimosine for 16 h. The treatment of the cycling cell population with 400 mM mimosine caused inhibition of DNA replication, changes in the progression of the cells in the cell cycle, and apoptosis. Nucleoid sedimentation analysis and comet assay were used to monitor the appearance and accumulation of DNA breaks. The rate of break accumulation was dose-dependent, did not depend on the stage of the cell cycle and was not connected with the mechanism of DNA replication. The data indicate that the effects of mimosine on DNA synthesis and the cell cycle may be a result of introduction of breaks into DNA. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Mimosine; DNA synthesis; Nucleoid sedimentation; Comet assay; Cell cycle
1. Introduction Mimosine, b-w N-Ž3-hydroxy-4-pyridone .x-aamino-propionic acid, is a rare nonprotein amino acid, derived from Mimosa and Leucaena plants and a reversible inhibitor of DNA synthesis. During the last few years, over a hundred papers appeared in which mimosine was used in procedures for the synchronization of mammalian cells at the G1rS
AbbreÕiations: dNTP, deoxynucleotide triphosphate; FACS, fluorescence activated cell sorting; MEL, mouse erythroleukemia; PBL, peripheral blood lymphocytes; RNR, ribonucleotide reductase; SHMT, serine hydroxymethyltransferase ) Corresponding author. Tel.: q359-2-739-954; fax: q359-2723-507. E-mail address: [email protected] ŽB. Anachkova..
phase boundary of the cell cycle. It was also suggested that being a specific cell cycle blocker, mimosine could be effective in designing new strategies for cancer therapy. In connection with this, a considerable effort has been made to study its mechanism of action. However, the diversity of the results obtained so far did not permit a single conclusion to be drawn neither in respect to its cell cycle blocking potential, nor to its effect on DNA replication. The first reports showed that mimosine reversibly arrested cycling human lymphoblastoid cells in G1 phase of the cell cycle at a point approximately 2 h prior to the G1rS phase transition as defined by aphidicolin w1x. Shortly after this paper appeared, it was reported that mimosine did not arrest cells in the late G1 phase, but rather, prevented the initiation of DNA synthesis at replication origins, and in addition,
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slowed down DNA chain elongation w2–5x. The authors had identified the enzyme serine hydroxymethyltransferase ŽSHMT. as a bonafide target for the drug and argued that mimosine executed its effect by binding to the enzyme. Meanwhile, it was demonstrated that mimosine inhibited DNA synthesis at the level of DNA chains elongation by causing a depletion of the intracellular pools of deoxynucleotide triphosphates ŽdNTPs. w6x. The authors did not explain this depletion with the binding of mimosine to SHMT, but with the inhibition of another enzyme playing a key role in dNTP metabolism, namely ribonucleotide reductase ŽRNR.. They argued that mimosine might disturb RNR function through its ability to chelate metal ions, since mimosine is a strong iron chelator and the enzyme has an essential requirement for iron for proper function. This hypothesis has been supported by the finding that mimosine arrests cycling cells only after they have entered the S phase w7x, and that mimosine did not have any effect on DNA synthesis in two DNA replication systems, in which DNA replication did not depend on de novo synthesis of dNTPs — in Xenopus embryonic cells w8x and in vitro replication of SV40 w9x. In a well-documented study, evidence was presented that the treatment of mammalian cells with mimosine led to the inhibition of a cyclin E-associated kinase and suggested the existence of a p53-independent, p21-dependent checkpoint pathway in late G1 phase of the cell cycle w10x. It was hypothesized that the activation of the checkpoint was a result of the introduction of DNA breaks due to the imbalance of the cellular dNTP pools caused by the inhibitory effect of mimosine on the enzymes SHMT and RNR. The hypothesis was based on literature data indicating that cells treated with mimosine underwent changes in their nuclear morphology and chromatin organization w11,12x and that mimosine has a clastogenic effect and can induce apoptosis w13x. In a recent paper w14x, we have shown that mimosine affects both replicon initiation and DNA chain elongation in a way similar to that of g-radiation and different from that of hydroxyurea, which is a typical RNR inhibitor. Bearing in mind that the primary effect of g-radiation was to produce breaks in cellular DNA, in the present communication, we studied the possibility that mimosine might produce DNA
breaks independently of the process of DNA synthesis. By using two different techniques, nucleoid sedimentation and comet assay, we show that the primary effect of mimosine is the introduction of breaks in chromosomal DNA.
2. Materials and methods 2.1. Cells and labeling Mouse erythroleukemia ŽMEL. cells, clone F4N, were cultured in MEM-S medium supplemented with 10% fetal calf serum. To determine the rate of DNA synthesis, aliquots of 2 = 10 6 exponentially growing cells were labeled with 1 mCirml w3 Hxthymidine for 30 min, DNA was precipitated onto Whatman GF-C filters with 10% ice-cold trichloracetic acid and the acid-soluble material washed away with 5% ice-cold trichloracetic acid. The filters were counted in a liquid scintillation counter and the specific radioactivity of DNA was calculated as cpmr100 cells. Human peripheral blood from healthy donors was obtained from the National Center for Blood Transfusion in Sofia. Peripheral blood lymphocytes ŽPBL. were purified twice by centrifugation through Ficoll layer at 500 = g for 20 min at room temperature. Cells were collected from the liquid interphase. PBL were washed with phosphate-buffered saline, pH 7.3 and resuspended to make 10 5 cellsrml in RPMI medium supplemented with 20% fetal calf serum. 2.2. Nucleoid sedimentation Nucleoid sedimentation was performed as described in Refs. w15,16x, using slightly different centrifugation conditions. Exponentially growing MEL cells were labeled overnight with 1 mCirml w3 Hxthymidine. The cells were collected by centrifugation at 1000 rpm, suspended in 1r10 volume of phosphate-buffered saline and 200 ml Žapproximately 5 = 10 5 cells. were applied on 200-ml lysis buffer Ž2 M NaCl, 1% Triton X100, 10 mM Tris–HCl, 10 mM EDTA, pH 8. layered on top of linear 10–30% sucrose density gradients prepared in 1 M NaCl, 5 mM Tris–HCl pH 8, 5 mM EDTA. Gradients were centrifuged in the Beckman SW 41 rotor at room temperature at 18 000 rpm for 30 min. They were
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fractionated by means of a peristaltic pump from the top and aliquots were taken and counted to determine the positions of the DNA peaks.
pH 8, and the gels were stained with ethidium bromide.
2.3. Comet assay
3. Results
Alkaline comet assay was performed essentially as described in Ref. w17x. The cells were suspended in low melting agarose Ž0.7% final concentration. and were poured on microscope slides, previously treated with 0.5% agarose. The cells were lysed in 1 M NaCl, 50 mM EDTA, 30 mM NaOH, 0.1% sodium laurylsarcosine and were electrophoresed in 30 mM NaOH, 10 mM EDTA at 0.5 Vrcm for 20 min. Gels were neutralized in 0.4 M Tris–HCl, pH 7.5, fixed in 70% ethanol and stained with ethidium bromide. Cells were examined with Leitz fluorescent microscope.
3.1. Effect of mimosine on DNA
2.4. Fluorescence actiÕated cell sorting (FACS) analysis FACS analysis was performed as described in Ref. w18x. Briefly, cells were pelleted, washed with phosphate buffered saline, treated with 20 mgrml RNase for 30 min at 378C and stained with 20 mgrml propidium iodide at room temperature for 30 min. 2 = 10 4 cellsrsample were analyzed with a Becton Dickinson ŽFacscalibur. cell sorter, using ModFit software ŽBecton Dickinson..
Exponentially growing MEL cells were treated with 50, 150 and 400 mM mimosine for 16 h. The duration of the treatment was chosen because 16 h was the approximate length of the division cycle of this cell line, and the highest concentration was the most common concentration of mimosine used for the synchronization of cell cultures at the G1rS boundary of the cell cycle. At the 2nd, 8th and 16th hours, aliquots were withdrawn for the labeling and determination of the specific radioactivity of DNA. The results showed that mimosine caused a well-expressed dose-dependent inhibition of DNA synthesis in MEL cells. When 400 mM mimosine was used, the inhibition of DNA synthesis was almost complete and was manifested in the first 2 h of treatment ŽFig. 1.. To monitor the DNA breaks formation, we applied two methods introduced and widely used for measuring the accumulation of single strand Žand
2.5. Isolation and electrophoresis of DNA To isolate genomic DNA, cells were lysed in 1 M NaCl, 0.5% sodium dodecylsulphate, 20 mM EDTA, 50 mM Tris–HCl, pH 8. Proteins were digested with 200 mgrml Proteinase K overnight, and after deproteinization with phenol–chloroform Ž1:1. and with chloroform–isoamyl alcohol Ž24:1., DNA was precipitated with two volumes of ethanol. It was dissolved in 10 mM Tris, 1 mM EDTA, pH 8 and RNA was degraded with 200 mgrml RNase at 378C for 3 h. The samples were deproteinized as above, precipitated with ethanol, and dissolved in 10 mM Tris, 1 mM EDTA, pH 8. DNA concentration was determined by reading the optical density at 260 nm. Agarose gel electrophoresis was performed in 1% agarose in 0.1 M Tris–acetic acid, 0.4 mM EDTA,
Fig. 1. Effect of mimosine on DNA synthesis. Exponentially growing MEL cells were treated with 50 mM Žv ., 150 mM ŽB. and 400 mM Ž'. mimosine for 2, 8 and 16 h. At the end of the periods, the cells were incubated with 1 mCirml w3 Hxthymidine for 30 min and the specific radioactivity of DNA determined. w3 Hxthymidine incorporation as a percentage of the control is plotted vs. time. Figures are means of three independent experiments. Standard deviations from the mean are shown with error bars.
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Fig. 2. Effect of mimosine concentration on nucleoid sedimentation velocity. Exponentially growing MEL cells were treated with 50, 150 and 400 mM mimosine for 16 h and subjected to nucleoid sedimentation analysis.
double strand. breaks in DNA caused by genotoxic agents such as g-radiation and chemical compounds w19–22x. In the first experiment, we applied the method of nucleoid sedimentation, which we have previously used to study the effect of g-radiation on DNA replication w23x. Upon gentle lysis of cells in the presence of 1 M NaCl, structures called nucleoids are formed. In the nucleoids containing intact DNA, the latter is supercoiled and upon sucrose gradient centrifugation, sediments, as a well-defined peak, close to the bottom of the gradient. When the cells are treated with an agent that introduces breaks into DNA, the supercoiled structure of the nucleoids is relaxed and they sediment more slowly. The relative sedimentation, determined as the ratio between the distances traveled by the treated and control nucleoids, reflects the degree of DNA relaxation due to the accumulation of breaks caused by the agent. MEL cells were treated with increasing concentrations of mimosine for 16 h and subjected to nucleoid sedimentation analysis. The results showed that the effect of mimosine on DNA supercoiling was similar to the effect of low doses of g-radiation w23x. After the treatment with mimosine, DNA was relaxed and the nucleoids sedimented more slowly. The slowing down of the sedimentation rate depended on mimosine concentration ŽFig. 2. and correlated well with the inhibition of DNA synthesis ŽFig. 1..
To confirm that mimosine caused breakage of DNA, we used the alkaline version of the single-cell gel electrophoresis known as comet assay Ž17. as an alternative method. The cells are embedded in agarose; the agarose is poured as a thin layer onto microscope slides and the gels are treated with high salt and alkali, electrophoresed and stained. The obtained structures are observed with a fluorescent microscope. Under these conditions, cells with intact DNA are seen as having a symmetric halo of DNA, while during the electrophoresis, the DNA fragments and relaxed loops resulting from strand breaks migrate toward the anode, providing a comet-like image. The extent of DNA damage in our experiments was measured by the length of the comet tails. The MEL cells began to develop tails at the 2nd hour of mimosine treatment. The tail lengths increased during the next 6 h and then reached a plateau. These results confirmed that treatment of MEL cells with mimosine caused a breakage of the cellular DNA. The effect of mimosine was dose-dependent and there was a good correlation between the rate of inhibition of the overall DNA synthesis ŽFig. 1. and the rate of break accumulation in DNA ŽFig. 3.. It should be noted that in the first experiment, the nucleoid peaks were homogeneous, which indicated that all cells might have been affected in the same way. One important advantage of the comet assay over nucleoid sedimentation and other methods for
Fig. 3. Alkaline comet assay. Exponentially growing MEL cells were treated with 50 mM Žv ., 150 mM ŽB. and 400 mM Ž'. mimosine for different periods of time and subjected to alkaline comet assay. Lengths of the comet tails are measured in micrometers. Figures are means of 100 measurements. Error bars show the standard deviations from the mean.
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the detection and evaluation of DNA damage is the possibility to examine the individual cells in a heterogeneous cell population. Its implication showed that the obtained comet populations were quite homogeneous for all mimosine concentrations and time points. Since we worked with asynchronous cell populations, this could mean that mimosine caused a breakage of the DNA molecules, regardless of the phase of the cell cycle and the metabolic activity of DNA. That is why it was important to examine the distribution of the cells in the different compartments of the cell cycle during the mimosine treatment. At the specified time points, samples were subjected to FACS analysis. The results showed that at the starting time point, about 30% of the cells were in G1, 60% in S and 10% in G2rM phases of the cell cycle. During the course of the treatment, the distribution of the cells in the cell cycle was affected by mimosine in a dose-dependent way. The two low doses used in this study did not show a pronounced effect on the cell cycle Žnot shown.. However, at the 16th hour of treatment, the highest dose of 400 mM mimosine caused a twofold increase of the cells in the G1 phase and a twofold decrease of the cells in the S and G2rM phases of the cell cycle, which indicated that mimosine caused a partial block of the cells in the G1 phase ŽTable 1.. To confirm that mimosine caused a breakage of the DNA molecules regardless of the phase of the cell cycle and the metabolic activity of DNA, we performed the following experiment. Human lym-
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phocytes from peripheral blood were isolated and treated with 400 mM mimosine. Since PBL represent an almost 100% G0rG1 phase cell population and neither synthesize DNA, nor divide unless stimulated, they were an ideal model to study the effect of mimosine on DNA, independent of DNA synthesis. At the specified time intervals of mimosine treatment, aliquots were taken and analyzed by alkaline comet assay and FACS analysis. The results showed that as in the case with cycling MEL cells, all quiescent PBL synchronously developed tails ŽFig. 4.. At the end of the experiment, there were no changes in the cell cycle distribution as revealed by FACS analysis, and no signs of apoptosis were seen. This unambiguously showed that the break formation was not connected with the process of DNA synthesis and confirmed our conclusion that mimosine inflicts breaks in cellular DNA independently of the metabolic activity of DNA. 3.2. Apoptosis As there were data that in lymphoid cell lines treatment with mimosine induced apoptosis w13x, we further studied the cell cycle dynamics after removal of the drug. After 16 h in 400 mM mimosine, the MEL cells were transferred into fresh medium without mimosine and grown for another 16 h, and at the specified time intervals, aliquots were taken for FACS analysis. The results showed that 2 h after the removal of mimosine, the cells arrested in G1 and S
Table 1 Effect of 400 mM mimosine on the cell cycle. Exponentially growing MEL cells were treated with 400 mM mimosine for 16 h. At the 2nd, 8th and 16th hours, aliquots were withdrawn and analyzed by FACS analysis. The cells were transferred in a fresh medium without mimosine, and at the specified time intervals, aliquots were withdrawn and analyzed by FACS analysis. The results are the means of three independent experiments. The standard deviations from the mean are shown Treatment with mimosine
0h 2h 8h 16 h 2-h chase 8-h chase 16-h chase a
Apoptosisa
Phase G1
S
G2 q M
29.6 " 2.2% 41.6 " 5.1% 55.9 " 4.1% 59.5 " 0.6% 17.8 " 1.2% 6.1 " 2.4% 12.1 " 4.2%
59.9 " 2.1% 53.4 " 6.4% 38.5 " 6.9% 35.9 " 0.6% 70.9 " 1.8% 48.3 " 3.1% 52.3 " 7.3%
10.5 " 1% 5 " 0.4% 5.6 " 0.6% 4.6 " 0.2% 11.3 " 0.6% 45.6 " 3.7% 35.6 " 6.6%
- 1% 1.5 " 0.1% 2.1 " 0.3% 2.7 " 0.3% 3 " 0.4% 21.4 " 1.5% 29.8 " 2.7%
The program used does not include apoptotic cells in the population of cycling cells when determining the percentage of cells in the different phases of the cell cycle.
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phase resumed their progression through the S phase and entered G2 phase of the cell cycle. At the 8th hour of mimosine chase, the amount of G2rM cells reached a maximum of 46% of the cycling population and then began to decrease. However, at the 16th hour after the release from the mimosine block, the amount of G2rM cells was still threefold higher than the percentage of G2rM cells in exponentially growing cells, which was an indication of a partial arrest of the cells in G2rM phase of the cell cycle. During this period, an accumulation of apoptotic cells, as identified by the FACS program, was observed. The percentage of apoptotic cells increased to reach 30%, 16 h after its release from the block and about 8 h after the beginning of the mitotic wave. To verify these cells as apoptotic, we applied another qualitative method for the detection of apoptosis. We isolated DNA and ran it on agarose gel electrophoresis. The pattern showed the typical for apoptotic cells oligosome ladder, which was a clear indication for the accumulation of apoptotic cells ŽFig. 5.. The presence of apoptotic cells was also identified by microscopic examination of the cells
Fig. 5. Agarose gel electrophoresis of DNA. ŽA. Marker Ž1 bp ladder.; ŽB. marker Ž100 bp ladder.; Ž1. DNA isolated from control cells, untreated with mimosine; Ž2. DNA isolated from cells treated for 16 h with 400 mM mimosine; Ž3. – Ž5. DNA from cells treated for 16 h with 400 mM mimosine and recovered for 2, 8 and 16 h, respectively.
with the characteristic membrane blebbing and by the comet assay with the movement of most of the DNA from the head into the tails of the comets Žnot shown..
4. Discussion
Fig. 4. Alkaline comet assay. Human PBL were treated for 16 h with 400 mM mimosine and subjected to alkaline comet assay. ŽA. Control cell, not treated with mimosine. ŽB. 16 h in 400 mM mimosine. Cells were viewed at =250 using fluorescent microscope.
There is a discrepancy in the results reported in the literature about the effect of mimosine on asynchronously proliferating cell populations and the problem of the fate of the cells after mimosine release is not very well studied. Thus, treatment with 200 mM mimosine of human lymphoblastoid cells for 16 h arrested the cells G1 phase w1x, while in experiments with Chinese hamster ovary cells, DNA synthesis was almost completely inhibited by all mimosine concentrations used Ž100–400 mM., but there were no changes in the distribution of the cells in the cell cycle even after prolonged treatment with the drug w3x. Recently, the problem was reinvesti-
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gated and it was shown on human cell lines that there was a dose-dependent inhibition of DNA synthesis and treatment with 500-mM mimosine for 24 h synchronized the cells in the G1 phase of the cell cycle w24x. Our results are consistent with the previous studies showing that mimosine reversibly inhibits DNA synthesis and arrests the cells in G1 phase and after its removal from the medium, mimosine can cause the arrest of the cells in G2rM phase and apoptosis w1,13,24,25x. As we have pointed out in Section 1, a coherent explanation of the multiple and diverse effects of mimosine on DNA synthesis and the cell cycle is still lacking. It has been reported that the primary target of mimosine was the enzyme RNR w6x, or SHMT w5x. Both enzymes play a role in the de novo dNTP synthesis and it has been suggested that the inhibition of either of them, or both, would lead to a depletion and imbalance of the cellular dNTP pools. According to the first hypothesis, depletion of the dNTP pools by inhibition of RNR would cause a stalling of the replication forks w6x. However, the effect of hydroxyurea, a typical RNR inhibitor that depletes the cellular dNTP pools, differs from that of mimosine. Hydroxyurea inhibits DNA chain elongation almost completely in a matter of minutes. The effect of mimosine is slower and more complex. Thus, in addition to the inhibition of DNA chain elongation, it also inhibits the initiation of DNA replication w14x. Moreover, unlike hydroxyurea, the action of mimosine is not restricted to the S phase, but extends to the other phases of the cell cycle and even to the next generations. Mimosine has a clastogenic effect, causes apoptosis and cell cycle re-entry following mimosine synchronization leads to elevated protein levels of the checkpoint regulators p53 and p21 w13,25x. According to the second hypothesis, the imbalance of the dNTP pools caused by the inhibition of SHMT and RNR leads to an accumulation of DNA breaks, which, on its part, triggers the cell response to mimosine w10x. However, it has been shown that mimosine does not inhibit the enzymatic activity of SHMT w5x and the accumulation of DNA breaks due to an imbalance of the dNTP pools is restricted to the S phase of the cell cycle w26x. This leaves open the question of how these breaks could affect cells in the G1 phase, which is upstream of the S phase. Our finding that mimosine introduced breaks in DNA of all cells of
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an exponentially growing MEL cell population and in quiescent PBL shows that in addition to a possible formation of DNA breaks during the S phase as a result of binding to SHMT and inhibition of RNR, mimosine induces DNA breaks independently of the cell cycle progression and the process of DNA synthesis. This fact and the results showing that the appearance and disappearance of the observed biological effects of mimosine, such as the inhibition of DNA synthesis, the G1 phase arrest, etc., either paralleled, or followed the accumulation of DNA breaks, indicate that the breakage of DNA is the primary effect of mimosine that triggers the cellular response. In a previous paper, we have reported that the effect of mimosine on cycling cell populations closely mimics the effect of g-radiation w14x. Similarly to mimosine, g-radiation slows down DNA synthesis w23,27–29x. It causes blocks at several checkpoints, the best studied being the ‘‘Restriction point’’ in G1 phase and checkpoint ‘‘Radiation’’ in G2 phase and two other recently identified checkpoints in the late G1 and late S phases, respectively w30,31x. The arrest at these checkpoints is temporary and in a few hours, the cells resume their progression through the cell cycle. Nevertheless, later on, most of the cells die by apoptosis w32,33x. g-Radiation causes a plethora of damages in DNA, most prominent of them being DNA breaks, which were identified as the primary signal for its biological effect w27–29,34x. The finding reported here that mimosine inflicts breaks in DNA regardless of the cell cycle phase and DNA synthesizing activity could explain why the effects of mimosine and g-radiation, two agents that have nothing in common except that both produce breaks in cellular DNA, are so similar. For the time being, we do not know what is the molecular mechanism of mimosine that induced the breakage of DNA. One possible mechanism is that mimosine, being a strong chelating agent binds iron and copper ions, and thus, facilitates the oxidative generation of free radicals. Mimosine has been widely used as a synchronizing agent in a number of studies of cell cycle control mechanisms and genome organization in mammalian cells. However, we would point out that mimosine should be avoided as a synchronizing agent in experiments in which the integrity of the isolated DNA is
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critical for the reliability of the results because of its DNA-cutting potential. Acknowledgements This work was supported by the Bulgarian National Research Fund Žgrant K-604r1997.. References w1x M. Lalande, A reversible arrest point in the late G1 phase of the mammalian cell cycle, Exp. Cell Res. 186 Ž1990. 332– 339. w2x P. Dijkwel, J. Hamlin, Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S-period in CHO cells synchronized with the plant amino acid mimosine, Mol. Cell. Biol. 12 Ž1992. 3715–3722. w3x P. Mosca, P. Dijkwel, J. Hamlin, The plant amino acid mimosine may inhibit initiation at origins of replication in Chinese hamster cells, Mol. Cell. Biol. 12 Ž1992. 4375–4383. w4x P. Mosca, H.-B. Lin, J. Hamlin, Mimosine, a novel inhibitor of DNA replication, binds to a 50 kDa protein in Chinese hamster cells, Nucl. Acids Res. 23 Ž1995. 261–268. w5x H.-B. Lin, R. Falchetto, P. Mosca, J. Shabanowitz, D. Hunts, J. Hamlin, Mimosine targets serine hydroxymethyltransferase, J. Biol. Chem. 271 Ž1996. 2548–2556. w6x D. Gilbert, A. Neilson, H. Miyazawa, M. DePamphilis, W. Burhans, Mimosine arrests DNA synthesis at replication forks by inhibiting deoxyribonucleotide metabolism, J. Biol. Chem. 270 Ž1995. 9597–9606. w7x T. Hughes, P. Cook, Mimosine arrests the cell cycle after cells enter S-phase, Exp. Cell Res. 222 Ž1996. 275–280. w8x Y. Wang, J. Zhao, J. Clapper, L.D. Martin, C. Du, E.R. DeVore, K. Harkins, D.L. Dobbs, R.M. Benbow, Mimosine differentially inhibits DNA replication and cell cycle progression in somatic cells compared to embryonic cells of Xenopus laeÕis, Exp. Cell Res. 217 Ž1995. 84–91. w9x R. Kalejta, J. Hamlin, The dual effect of mimosine on DNA replication, Exp. Cell Res. 231 Ž1997. 173–183. w10x R. Alpan, A. Pardee, p21WA F1rCIP1SDI1 is elevated through a p53-independent pathway by mimosine, Cell Growth Differ. 7 Ž1996. 893–901. w11x G. Vogt, In vivo decondensation of chromatin and nuclear fibrillar component by Leucaena leucocephala ingredient, Biol. Cell 72 Ž1991. 211–215. w12x G. Vogt, R. Bohm, H. Segner, Mimosine, a naturally occurring drug interfering primarily with the cell nucleus, J. Submicrosc. Cytol. Pathol. 25 Ž1993. 247–256. w13x A.N. Jha, P.M. Hande, L.H.F. Mullenders, A.T. Natarajan, Mimosine is a potent clastogen in primary and transformed hamster fibroblasts but not in primary and transformed human lymphocytes, Mutagenesis 10 Ž1995. 385–391. w14x L. Tsvetkov, G. Russev, B. Anachkova, Effect of mimosine on DNA synthesis in mammalian cells, Cancer Res. 57 Ž1997. 2252–2255.
w15x P.R. Cook, J.A. Brazel, Supercoils in human DNA, J. Cell Sci. 19 Ž1975. 261–279. w16x M. Walicka, E. Godlewska, Radiation sensitivities are not related to the size of DNA supercoiled domains in L5178Y-R and L5178Y-S cells, Int. J. Radiat. Biol. 55 Ž1989. 953–961. w17x P.L. Olive, J.P. Banath, C.D. Fjell, DNA strand breakage and DNA structure influence staining with propidium iodide using the alkaline comet assay, Cytometry 16 Ž1994. 305–312. w18x J. Watson, E. Erba, Flow cytometry, in: R. Freshney ŽEd.., Animal Cell Culture, IRL Press, Oxford, 1992, pp. 165–213. w19x P.R. Cook, I. Brazel, Detection and repair of single strand breaks in nuclear DNA, Nature 263 Ž1976. 679–682. w20x O. Ostling, K.J. Johanson, Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells, Biochim. Biophys. Res. Commun. 123 Ž1984. 291–298. w21x D.W. Fairbairn, P.L. Olive, K.L. O’Neill, The comet assay: a comprehensive review, Mutat. Res. 339 Ž1995. 37–59. w22x D. Anderson, T.-W. Yu, D.B. McGregor, Comet assay responses as indicators of carcinogen exposure, Mutagenesis 13 Ž1998. 539–555. w23x D. Kunnev, L. Tsvetkov, B. Anachkova, G. Russev, Clusters of replicons that fire simultaneously may be organized into superloops, DNA Cell Biol. 16 Ž1997. 1059–1065. w24x T. Krude, Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner, Exp. Cell Res. 247 Ž1999. 148–159. w25x O. Ji, L.J. Marnett, J.A. Pietenpol, Cell cycle re-entry following chemically induced synchronization leads to elevated p53 and p21 protein levels, Oncogene 15 Ž1997. 2749–2753. w26x A. Yoshioka, S. Tanaka, O. Hiraoka, Y. Koyama, Y. Hirota, D. Ayusawa, T. Seno, C. Garrett, Y. Wataya, Deoxyribonucleoside triphosphate imbalance, J. Biol. Chem. 262 Ž1987. 8235–8241. w27x R.B. Painter, R.B. Young, Radiosensitivity in ataxia– telangiectasia: a new explanation, Proc. Natl. Acad. Sci. U. S. A. 77 Ž1980. 7315–7317. w28x R.B. Painter, Inhibition of mammalian cell DNA synthesis by ionizing radiation, Int. J. Radiat. Biol. 49 Ž1986. 771–781. w29x A. Lallev, B. Anachkova, G. Russev, Effect of ionizing radiation and topoisomerase II inhibitors on DNA synthesis in mammalian cells, Eur. J. Biochem. 216 Ž1993. 177–181. w30x J.A. D’Anna, J.G. Valdez, R.C. Habbersett, H.A. Crissman, Association of G1rS-phase and late S-phase checkpoints with regulation of cyclin-dependent kinases in Chinese hamster ovary cells, Radiat. Res. 148 Ž197. 260–271. w31x J.M. Larner, H. Lee, J.L.S. Hamlin, S phase damage sensing checkpoints in mammalian cells, Cancer Surv. 29 Ž1997. 25–45. w32x J.S. Bedford, Sublethal damage, potentially lethal damage, and chromosomal aberrations in mammalian cells exposed to ionizing radiation, Int. J. Radiat. Oncol., Biol., Phys. 21 Ž1991. 37–42. w33x T.J. McMillan, J.K. Peacock, Molecular determinants of radiosensitivity in mammalian cells, Int. J. Radiat. Biol. 65 Ž1994. 49–55. w34x D.T. Goodhead, Initial events in the cellular effects of ionizing radiations: clustered damage in DNA, Int. J. Radiat. Biol. 65 Ž1994. 7–17.
Treatment of mammalian cells with mimosine generates DNA breaks Ivailo Mikhailov, George Russev, Boyka Anachkova) Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. BoncheÕ Street, Bl. 21, 1113 Sofia, Bulgaria Received 6 September 1999; received in revised form 4 February 2000; accepted 4 February 2000
Abstract Exponentially growing mouse erythroleukemia ŽMEL. cells and quiescent human peripheral blood lymphocytes ŽPBL. were treated with different concentrations of the nonprotein amino acid mimosine for 16 h. The treatment of the cycling cell population with 400 mM mimosine caused inhibition of DNA replication, changes in the progression of the cells in the cell cycle, and apoptosis. Nucleoid sedimentation analysis and comet assay were used to monitor the appearance and accumulation of DNA breaks. The rate of break accumulation was dose-dependent, did not depend on the stage of the cell cycle and was not connected with the mechanism of DNA replication. The data indicate that the effects of mimosine on DNA synthesis and the cell cycle may be a result of introduction of breaks into DNA. q 2000 Elsevier Science B.V. All rights reserved. Keywords: Mimosine; DNA synthesis; Nucleoid sedimentation; Comet assay; Cell cycle
1. Introduction Mimosine, b-w N-Ž3-hydroxy-4-pyridone .x-aamino-propionic acid, is a rare nonprotein amino acid, derived from Mimosa and Leucaena plants and a reversible inhibitor of DNA synthesis. During the last few years, over a hundred papers appeared in which mimosine was used in procedures for the synchronization of mammalian cells at the G1rS
AbbreÕiations: dNTP, deoxynucleotide triphosphate; FACS, fluorescence activated cell sorting; MEL, mouse erythroleukemia; PBL, peripheral blood lymphocytes; RNR, ribonucleotide reductase; SHMT, serine hydroxymethyltransferase ) Corresponding author. Tel.: q359-2-739-954; fax: q359-2723-507. E-mail address: [email protected] ŽB. Anachkova..
phase boundary of the cell cycle. It was also suggested that being a specific cell cycle blocker, mimosine could be effective in designing new strategies for cancer therapy. In connection with this, a considerable effort has been made to study its mechanism of action. However, the diversity of the results obtained so far did not permit a single conclusion to be drawn neither in respect to its cell cycle blocking potential, nor to its effect on DNA replication. The first reports showed that mimosine reversibly arrested cycling human lymphoblastoid cells in G1 phase of the cell cycle at a point approximately 2 h prior to the G1rS phase transition as defined by aphidicolin w1x. Shortly after this paper appeared, it was reported that mimosine did not arrest cells in the late G1 phase, but rather, prevented the initiation of DNA synthesis at replication origins, and in addition,
0921-8777r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 1 - 8 7 7 7 Ž 0 0 . 0 0 0 0 7 - 0
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slowed down DNA chain elongation w2–5x. The authors had identified the enzyme serine hydroxymethyltransferase ŽSHMT. as a bonafide target for the drug and argued that mimosine executed its effect by binding to the enzyme. Meanwhile, it was demonstrated that mimosine inhibited DNA synthesis at the level of DNA chains elongation by causing a depletion of the intracellular pools of deoxynucleotide triphosphates ŽdNTPs. w6x. The authors did not explain this depletion with the binding of mimosine to SHMT, but with the inhibition of another enzyme playing a key role in dNTP metabolism, namely ribonucleotide reductase ŽRNR.. They argued that mimosine might disturb RNR function through its ability to chelate metal ions, since mimosine is a strong iron chelator and the enzyme has an essential requirement for iron for proper function. This hypothesis has been supported by the finding that mimosine arrests cycling cells only after they have entered the S phase w7x, and that mimosine did not have any effect on DNA synthesis in two DNA replication systems, in which DNA replication did not depend on de novo synthesis of dNTPs — in Xenopus embryonic cells w8x and in vitro replication of SV40 w9x. In a well-documented study, evidence was presented that the treatment of mammalian cells with mimosine led to the inhibition of a cyclin E-associated kinase and suggested the existence of a p53-independent, p21-dependent checkpoint pathway in late G1 phase of the cell cycle w10x. It was hypothesized that the activation of the checkpoint was a result of the introduction of DNA breaks due to the imbalance of the cellular dNTP pools caused by the inhibitory effect of mimosine on the enzymes SHMT and RNR. The hypothesis was based on literature data indicating that cells treated with mimosine underwent changes in their nuclear morphology and chromatin organization w11,12x and that mimosine has a clastogenic effect and can induce apoptosis w13x. In a recent paper w14x, we have shown that mimosine affects both replicon initiation and DNA chain elongation in a way similar to that of g-radiation and different from that of hydroxyurea, which is a typical RNR inhibitor. Bearing in mind that the primary effect of g-radiation was to produce breaks in cellular DNA, in the present communication, we studied the possibility that mimosine might produce DNA
breaks independently of the process of DNA synthesis. By using two different techniques, nucleoid sedimentation and comet assay, we show that the primary effect of mimosine is the introduction of breaks in chromosomal DNA.
2. Materials and methods 2.1. Cells and labeling Mouse erythroleukemia ŽMEL. cells, clone F4N, were cultured in MEM-S medium supplemented with 10% fetal calf serum. To determine the rate of DNA synthesis, aliquots of 2 = 10 6 exponentially growing cells were labeled with 1 mCirml w3 Hxthymidine for 30 min, DNA was precipitated onto Whatman GF-C filters with 10% ice-cold trichloracetic acid and the acid-soluble material washed away with 5% ice-cold trichloracetic acid. The filters were counted in a liquid scintillation counter and the specific radioactivity of DNA was calculated as cpmr100 cells. Human peripheral blood from healthy donors was obtained from the National Center for Blood Transfusion in Sofia. Peripheral blood lymphocytes ŽPBL. were purified twice by centrifugation through Ficoll layer at 500 = g for 20 min at room temperature. Cells were collected from the liquid interphase. PBL were washed with phosphate-buffered saline, pH 7.3 and resuspended to make 10 5 cellsrml in RPMI medium supplemented with 20% fetal calf serum. 2.2. Nucleoid sedimentation Nucleoid sedimentation was performed as described in Refs. w15,16x, using slightly different centrifugation conditions. Exponentially growing MEL cells were labeled overnight with 1 mCirml w3 Hxthymidine. The cells were collected by centrifugation at 1000 rpm, suspended in 1r10 volume of phosphate-buffered saline and 200 ml Žapproximately 5 = 10 5 cells. were applied on 200-ml lysis buffer Ž2 M NaCl, 1% Triton X100, 10 mM Tris–HCl, 10 mM EDTA, pH 8. layered on top of linear 10–30% sucrose density gradients prepared in 1 M NaCl, 5 mM Tris–HCl pH 8, 5 mM EDTA. Gradients were centrifuged in the Beckman SW 41 rotor at room temperature at 18 000 rpm for 30 min. They were
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fractionated by means of a peristaltic pump from the top and aliquots were taken and counted to determine the positions of the DNA peaks.
pH 8, and the gels were stained with ethidium bromide.
2.3. Comet assay
3. Results
Alkaline comet assay was performed essentially as described in Ref. w17x. The cells were suspended in low melting agarose Ž0.7% final concentration. and were poured on microscope slides, previously treated with 0.5% agarose. The cells were lysed in 1 M NaCl, 50 mM EDTA, 30 mM NaOH, 0.1% sodium laurylsarcosine and were electrophoresed in 30 mM NaOH, 10 mM EDTA at 0.5 Vrcm for 20 min. Gels were neutralized in 0.4 M Tris–HCl, pH 7.5, fixed in 70% ethanol and stained with ethidium bromide. Cells were examined with Leitz fluorescent microscope.
3.1. Effect of mimosine on DNA
2.4. Fluorescence actiÕated cell sorting (FACS) analysis FACS analysis was performed as described in Ref. w18x. Briefly, cells were pelleted, washed with phosphate buffered saline, treated with 20 mgrml RNase for 30 min at 378C and stained with 20 mgrml propidium iodide at room temperature for 30 min. 2 = 10 4 cellsrsample were analyzed with a Becton Dickinson ŽFacscalibur. cell sorter, using ModFit software ŽBecton Dickinson..
Exponentially growing MEL cells were treated with 50, 150 and 400 mM mimosine for 16 h. The duration of the treatment was chosen because 16 h was the approximate length of the division cycle of this cell line, and the highest concentration was the most common concentration of mimosine used for the synchronization of cell cultures at the G1rS boundary of the cell cycle. At the 2nd, 8th and 16th hours, aliquots were withdrawn for the labeling and determination of the specific radioactivity of DNA. The results showed that mimosine caused a well-expressed dose-dependent inhibition of DNA synthesis in MEL cells. When 400 mM mimosine was used, the inhibition of DNA synthesis was almost complete and was manifested in the first 2 h of treatment ŽFig. 1.. To monitor the DNA breaks formation, we applied two methods introduced and widely used for measuring the accumulation of single strand Žand
2.5. Isolation and electrophoresis of DNA To isolate genomic DNA, cells were lysed in 1 M NaCl, 0.5% sodium dodecylsulphate, 20 mM EDTA, 50 mM Tris–HCl, pH 8. Proteins were digested with 200 mgrml Proteinase K overnight, and after deproteinization with phenol–chloroform Ž1:1. and with chloroform–isoamyl alcohol Ž24:1., DNA was precipitated with two volumes of ethanol. It was dissolved in 10 mM Tris, 1 mM EDTA, pH 8 and RNA was degraded with 200 mgrml RNase at 378C for 3 h. The samples were deproteinized as above, precipitated with ethanol, and dissolved in 10 mM Tris, 1 mM EDTA, pH 8. DNA concentration was determined by reading the optical density at 260 nm. Agarose gel electrophoresis was performed in 1% agarose in 0.1 M Tris–acetic acid, 0.4 mM EDTA,
Fig. 1. Effect of mimosine on DNA synthesis. Exponentially growing MEL cells were treated with 50 mM Žv ., 150 mM ŽB. and 400 mM Ž'. mimosine for 2, 8 and 16 h. At the end of the periods, the cells were incubated with 1 mCirml w3 Hxthymidine for 30 min and the specific radioactivity of DNA determined. w3 Hxthymidine incorporation as a percentage of the control is plotted vs. time. Figures are means of three independent experiments. Standard deviations from the mean are shown with error bars.
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Fig. 2. Effect of mimosine concentration on nucleoid sedimentation velocity. Exponentially growing MEL cells were treated with 50, 150 and 400 mM mimosine for 16 h and subjected to nucleoid sedimentation analysis.
double strand. breaks in DNA caused by genotoxic agents such as g-radiation and chemical compounds w19–22x. In the first experiment, we applied the method of nucleoid sedimentation, which we have previously used to study the effect of g-radiation on DNA replication w23x. Upon gentle lysis of cells in the presence of 1 M NaCl, structures called nucleoids are formed. In the nucleoids containing intact DNA, the latter is supercoiled and upon sucrose gradient centrifugation, sediments, as a well-defined peak, close to the bottom of the gradient. When the cells are treated with an agent that introduces breaks into DNA, the supercoiled structure of the nucleoids is relaxed and they sediment more slowly. The relative sedimentation, determined as the ratio between the distances traveled by the treated and control nucleoids, reflects the degree of DNA relaxation due to the accumulation of breaks caused by the agent. MEL cells were treated with increasing concentrations of mimosine for 16 h and subjected to nucleoid sedimentation analysis. The results showed that the effect of mimosine on DNA supercoiling was similar to the effect of low doses of g-radiation w23x. After the treatment with mimosine, DNA was relaxed and the nucleoids sedimented more slowly. The slowing down of the sedimentation rate depended on mimosine concentration ŽFig. 2. and correlated well with the inhibition of DNA synthesis ŽFig. 1..
To confirm that mimosine caused breakage of DNA, we used the alkaline version of the single-cell gel electrophoresis known as comet assay Ž17. as an alternative method. The cells are embedded in agarose; the agarose is poured as a thin layer onto microscope slides and the gels are treated with high salt and alkali, electrophoresed and stained. The obtained structures are observed with a fluorescent microscope. Under these conditions, cells with intact DNA are seen as having a symmetric halo of DNA, while during the electrophoresis, the DNA fragments and relaxed loops resulting from strand breaks migrate toward the anode, providing a comet-like image. The extent of DNA damage in our experiments was measured by the length of the comet tails. The MEL cells began to develop tails at the 2nd hour of mimosine treatment. The tail lengths increased during the next 6 h and then reached a plateau. These results confirmed that treatment of MEL cells with mimosine caused a breakage of the cellular DNA. The effect of mimosine was dose-dependent and there was a good correlation between the rate of inhibition of the overall DNA synthesis ŽFig. 1. and the rate of break accumulation in DNA ŽFig. 3.. It should be noted that in the first experiment, the nucleoid peaks were homogeneous, which indicated that all cells might have been affected in the same way. One important advantage of the comet assay over nucleoid sedimentation and other methods for
Fig. 3. Alkaline comet assay. Exponentially growing MEL cells were treated with 50 mM Žv ., 150 mM ŽB. and 400 mM Ž'. mimosine for different periods of time and subjected to alkaline comet assay. Lengths of the comet tails are measured in micrometers. Figures are means of 100 measurements. Error bars show the standard deviations from the mean.
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the detection and evaluation of DNA damage is the possibility to examine the individual cells in a heterogeneous cell population. Its implication showed that the obtained comet populations were quite homogeneous for all mimosine concentrations and time points. Since we worked with asynchronous cell populations, this could mean that mimosine caused a breakage of the DNA molecules, regardless of the phase of the cell cycle and the metabolic activity of DNA. That is why it was important to examine the distribution of the cells in the different compartments of the cell cycle during the mimosine treatment. At the specified time points, samples were subjected to FACS analysis. The results showed that at the starting time point, about 30% of the cells were in G1, 60% in S and 10% in G2rM phases of the cell cycle. During the course of the treatment, the distribution of the cells in the cell cycle was affected by mimosine in a dose-dependent way. The two low doses used in this study did not show a pronounced effect on the cell cycle Žnot shown.. However, at the 16th hour of treatment, the highest dose of 400 mM mimosine caused a twofold increase of the cells in the G1 phase and a twofold decrease of the cells in the S and G2rM phases of the cell cycle, which indicated that mimosine caused a partial block of the cells in the G1 phase ŽTable 1.. To confirm that mimosine caused a breakage of the DNA molecules regardless of the phase of the cell cycle and the metabolic activity of DNA, we performed the following experiment. Human lym-
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phocytes from peripheral blood were isolated and treated with 400 mM mimosine. Since PBL represent an almost 100% G0rG1 phase cell population and neither synthesize DNA, nor divide unless stimulated, they were an ideal model to study the effect of mimosine on DNA, independent of DNA synthesis. At the specified time intervals of mimosine treatment, aliquots were taken and analyzed by alkaline comet assay and FACS analysis. The results showed that as in the case with cycling MEL cells, all quiescent PBL synchronously developed tails ŽFig. 4.. At the end of the experiment, there were no changes in the cell cycle distribution as revealed by FACS analysis, and no signs of apoptosis were seen. This unambiguously showed that the break formation was not connected with the process of DNA synthesis and confirmed our conclusion that mimosine inflicts breaks in cellular DNA independently of the metabolic activity of DNA. 3.2. Apoptosis As there were data that in lymphoid cell lines treatment with mimosine induced apoptosis w13x, we further studied the cell cycle dynamics after removal of the drug. After 16 h in 400 mM mimosine, the MEL cells were transferred into fresh medium without mimosine and grown for another 16 h, and at the specified time intervals, aliquots were taken for FACS analysis. The results showed that 2 h after the removal of mimosine, the cells arrested in G1 and S
Table 1 Effect of 400 mM mimosine on the cell cycle. Exponentially growing MEL cells were treated with 400 mM mimosine for 16 h. At the 2nd, 8th and 16th hours, aliquots were withdrawn and analyzed by FACS analysis. The cells were transferred in a fresh medium without mimosine, and at the specified time intervals, aliquots were withdrawn and analyzed by FACS analysis. The results are the means of three independent experiments. The standard deviations from the mean are shown Treatment with mimosine
0h 2h 8h 16 h 2-h chase 8-h chase 16-h chase a
Apoptosisa
Phase G1
S
G2 q M
29.6 " 2.2% 41.6 " 5.1% 55.9 " 4.1% 59.5 " 0.6% 17.8 " 1.2% 6.1 " 2.4% 12.1 " 4.2%
59.9 " 2.1% 53.4 " 6.4% 38.5 " 6.9% 35.9 " 0.6% 70.9 " 1.8% 48.3 " 3.1% 52.3 " 7.3%
10.5 " 1% 5 " 0.4% 5.6 " 0.6% 4.6 " 0.2% 11.3 " 0.6% 45.6 " 3.7% 35.6 " 6.6%
- 1% 1.5 " 0.1% 2.1 " 0.3% 2.7 " 0.3% 3 " 0.4% 21.4 " 1.5% 29.8 " 2.7%
The program used does not include apoptotic cells in the population of cycling cells when determining the percentage of cells in the different phases of the cell cycle.
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phase resumed their progression through the S phase and entered G2 phase of the cell cycle. At the 8th hour of mimosine chase, the amount of G2rM cells reached a maximum of 46% of the cycling population and then began to decrease. However, at the 16th hour after the release from the mimosine block, the amount of G2rM cells was still threefold higher than the percentage of G2rM cells in exponentially growing cells, which was an indication of a partial arrest of the cells in G2rM phase of the cell cycle. During this period, an accumulation of apoptotic cells, as identified by the FACS program, was observed. The percentage of apoptotic cells increased to reach 30%, 16 h after its release from the block and about 8 h after the beginning of the mitotic wave. To verify these cells as apoptotic, we applied another qualitative method for the detection of apoptosis. We isolated DNA and ran it on agarose gel electrophoresis. The pattern showed the typical for apoptotic cells oligosome ladder, which was a clear indication for the accumulation of apoptotic cells ŽFig. 5.. The presence of apoptotic cells was also identified by microscopic examination of the cells
Fig. 5. Agarose gel electrophoresis of DNA. ŽA. Marker Ž1 bp ladder.; ŽB. marker Ž100 bp ladder.; Ž1. DNA isolated from control cells, untreated with mimosine; Ž2. DNA isolated from cells treated for 16 h with 400 mM mimosine; Ž3. – Ž5. DNA from cells treated for 16 h with 400 mM mimosine and recovered for 2, 8 and 16 h, respectively.
with the characteristic membrane blebbing and by the comet assay with the movement of most of the DNA from the head into the tails of the comets Žnot shown..
4. Discussion
Fig. 4. Alkaline comet assay. Human PBL were treated for 16 h with 400 mM mimosine and subjected to alkaline comet assay. ŽA. Control cell, not treated with mimosine. ŽB. 16 h in 400 mM mimosine. Cells were viewed at =250 using fluorescent microscope.
There is a discrepancy in the results reported in the literature about the effect of mimosine on asynchronously proliferating cell populations and the problem of the fate of the cells after mimosine release is not very well studied. Thus, treatment with 200 mM mimosine of human lymphoblastoid cells for 16 h arrested the cells G1 phase w1x, while in experiments with Chinese hamster ovary cells, DNA synthesis was almost completely inhibited by all mimosine concentrations used Ž100–400 mM., but there were no changes in the distribution of the cells in the cell cycle even after prolonged treatment with the drug w3x. Recently, the problem was reinvesti-
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gated and it was shown on human cell lines that there was a dose-dependent inhibition of DNA synthesis and treatment with 500-mM mimosine for 24 h synchronized the cells in the G1 phase of the cell cycle w24x. Our results are consistent with the previous studies showing that mimosine reversibly inhibits DNA synthesis and arrests the cells in G1 phase and after its removal from the medium, mimosine can cause the arrest of the cells in G2rM phase and apoptosis w1,13,24,25x. As we have pointed out in Section 1, a coherent explanation of the multiple and diverse effects of mimosine on DNA synthesis and the cell cycle is still lacking. It has been reported that the primary target of mimosine was the enzyme RNR w6x, or SHMT w5x. Both enzymes play a role in the de novo dNTP synthesis and it has been suggested that the inhibition of either of them, or both, would lead to a depletion and imbalance of the cellular dNTP pools. According to the first hypothesis, depletion of the dNTP pools by inhibition of RNR would cause a stalling of the replication forks w6x. However, the effect of hydroxyurea, a typical RNR inhibitor that depletes the cellular dNTP pools, differs from that of mimosine. Hydroxyurea inhibits DNA chain elongation almost completely in a matter of minutes. The effect of mimosine is slower and more complex. Thus, in addition to the inhibition of DNA chain elongation, it also inhibits the initiation of DNA replication w14x. Moreover, unlike hydroxyurea, the action of mimosine is not restricted to the S phase, but extends to the other phases of the cell cycle and even to the next generations. Mimosine has a clastogenic effect, causes apoptosis and cell cycle re-entry following mimosine synchronization leads to elevated protein levels of the checkpoint regulators p53 and p21 w13,25x. According to the second hypothesis, the imbalance of the dNTP pools caused by the inhibition of SHMT and RNR leads to an accumulation of DNA breaks, which, on its part, triggers the cell response to mimosine w10x. However, it has been shown that mimosine does not inhibit the enzymatic activity of SHMT w5x and the accumulation of DNA breaks due to an imbalance of the dNTP pools is restricted to the S phase of the cell cycle w26x. This leaves open the question of how these breaks could affect cells in the G1 phase, which is upstream of the S phase. Our finding that mimosine introduced breaks in DNA of all cells of
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an exponentially growing MEL cell population and in quiescent PBL shows that in addition to a possible formation of DNA breaks during the S phase as a result of binding to SHMT and inhibition of RNR, mimosine induces DNA breaks independently of the cell cycle progression and the process of DNA synthesis. This fact and the results showing that the appearance and disappearance of the observed biological effects of mimosine, such as the inhibition of DNA synthesis, the G1 phase arrest, etc., either paralleled, or followed the accumulation of DNA breaks, indicate that the breakage of DNA is the primary effect of mimosine that triggers the cellular response. In a previous paper, we have reported that the effect of mimosine on cycling cell populations closely mimics the effect of g-radiation w14x. Similarly to mimosine, g-radiation slows down DNA synthesis w23,27–29x. It causes blocks at several checkpoints, the best studied being the ‘‘Restriction point’’ in G1 phase and checkpoint ‘‘Radiation’’ in G2 phase and two other recently identified checkpoints in the late G1 and late S phases, respectively w30,31x. The arrest at these checkpoints is temporary and in a few hours, the cells resume their progression through the cell cycle. Nevertheless, later on, most of the cells die by apoptosis w32,33x. g-Radiation causes a plethora of damages in DNA, most prominent of them being DNA breaks, which were identified as the primary signal for its biological effect w27–29,34x. The finding reported here that mimosine inflicts breaks in DNA regardless of the cell cycle phase and DNA synthesizing activity could explain why the effects of mimosine and g-radiation, two agents that have nothing in common except that both produce breaks in cellular DNA, are so similar. For the time being, we do not know what is the molecular mechanism of mimosine that induced the breakage of DNA. One possible mechanism is that mimosine, being a strong chelating agent binds iron and copper ions, and thus, facilitates the oxidative generation of free radicals. Mimosine has been widely used as a synchronizing agent in a number of studies of cell cycle control mechanisms and genome organization in mammalian cells. However, we would point out that mimosine should be avoided as a synchronizing agent in experiments in which the integrity of the isolated DNA is
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critical for the reliability of the results because of its DNA-cutting potential. Acknowledgements This work was supported by the Bulgarian National Research Fund Žgrant K-604r1997.. References w1x M. Lalande, A reversible arrest point in the late G1 phase of the mammalian cell cycle, Exp. Cell Res. 186 Ž1990. 332– 339. w2x P. Dijkwel, J. Hamlin, Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S-period in CHO cells synchronized with the plant amino acid mimosine, Mol. Cell. Biol. 12 Ž1992. 3715–3722. w3x P. Mosca, P. Dijkwel, J. Hamlin, The plant amino acid mimosine may inhibit initiation at origins of replication in Chinese hamster cells, Mol. Cell. Biol. 12 Ž1992. 4375–4383. w4x P. Mosca, H.-B. Lin, J. Hamlin, Mimosine, a novel inhibitor of DNA replication, binds to a 50 kDa protein in Chinese hamster cells, Nucl. Acids Res. 23 Ž1995. 261–268. w5x H.-B. Lin, R. Falchetto, P. Mosca, J. Shabanowitz, D. Hunts, J. Hamlin, Mimosine targets serine hydroxymethyltransferase, J. Biol. Chem. 271 Ž1996. 2548–2556. w6x D. Gilbert, A. Neilson, H. Miyazawa, M. DePamphilis, W. Burhans, Mimosine arrests DNA synthesis at replication forks by inhibiting deoxyribonucleotide metabolism, J. Biol. Chem. 270 Ž1995. 9597–9606. w7x T. Hughes, P. Cook, Mimosine arrests the cell cycle after cells enter S-phase, Exp. Cell Res. 222 Ž1996. 275–280. w8x Y. Wang, J. Zhao, J. Clapper, L.D. Martin, C. Du, E.R. DeVore, K. Harkins, D.L. Dobbs, R.M. Benbow, Mimosine differentially inhibits DNA replication and cell cycle progression in somatic cells compared to embryonic cells of Xenopus laeÕis, Exp. Cell Res. 217 Ž1995. 84–91. w9x R. Kalejta, J. Hamlin, The dual effect of mimosine on DNA replication, Exp. Cell Res. 231 Ž1997. 173–183. w10x R. Alpan, A. Pardee, p21WA F1rCIP1SDI1 is elevated through a p53-independent pathway by mimosine, Cell Growth Differ. 7 Ž1996. 893–901. w11x G. Vogt, In vivo decondensation of chromatin and nuclear fibrillar component by Leucaena leucocephala ingredient, Biol. Cell 72 Ž1991. 211–215. w12x G. Vogt, R. Bohm, H. Segner, Mimosine, a naturally occurring drug interfering primarily with the cell nucleus, J. Submicrosc. Cytol. Pathol. 25 Ž1993. 247–256. w13x A.N. Jha, P.M. Hande, L.H.F. Mullenders, A.T. Natarajan, Mimosine is a potent clastogen in primary and transformed hamster fibroblasts but not in primary and transformed human lymphocytes, Mutagenesis 10 Ž1995. 385–391. w14x L. Tsvetkov, G. Russev, B. Anachkova, Effect of mimosine on DNA synthesis in mammalian cells, Cancer Res. 57 Ž1997. 2252–2255.
w15x P.R. Cook, J.A. Brazel, Supercoils in human DNA, J. Cell Sci. 19 Ž1975. 261–279. w16x M. Walicka, E. Godlewska, Radiation sensitivities are not related to the size of DNA supercoiled domains in L5178Y-R and L5178Y-S cells, Int. J. Radiat. Biol. 55 Ž1989. 953–961. w17x P.L. Olive, J.P. Banath, C.D. Fjell, DNA strand breakage and DNA structure influence staining with propidium iodide using the alkaline comet assay, Cytometry 16 Ž1994. 305–312. w18x J. Watson, E. Erba, Flow cytometry, in: R. Freshney ŽEd.., Animal Cell Culture, IRL Press, Oxford, 1992, pp. 165–213. w19x P.R. Cook, I. Brazel, Detection and repair of single strand breaks in nuclear DNA, Nature 263 Ž1976. 679–682. w20x O. Ostling, K.J. Johanson, Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells, Biochim. Biophys. Res. Commun. 123 Ž1984. 291–298. w21x D.W. Fairbairn, P.L. Olive, K.L. O’Neill, The comet assay: a comprehensive review, Mutat. Res. 339 Ž1995. 37–59. w22x D. Anderson, T.-W. Yu, D.B. McGregor, Comet assay responses as indicators of carcinogen exposure, Mutagenesis 13 Ž1998. 539–555. w23x D. Kunnev, L. Tsvetkov, B. Anachkova, G. Russev, Clusters of replicons that fire simultaneously may be organized into superloops, DNA Cell Biol. 16 Ž1997. 1059–1065. w24x T. Krude, Mimosine arrests proliferating human cells before onset of DNA replication in a dose-dependent manner, Exp. Cell Res. 247 Ž1999. 148–159. w25x O. Ji, L.J. Marnett, J.A. Pietenpol, Cell cycle re-entry following chemically induced synchronization leads to elevated p53 and p21 protein levels, Oncogene 15 Ž1997. 2749–2753. w26x A. Yoshioka, S. Tanaka, O. Hiraoka, Y. Koyama, Y. Hirota, D. Ayusawa, T. Seno, C. Garrett, Y. Wataya, Deoxyribonucleoside triphosphate imbalance, J. Biol. Chem. 262 Ž1987. 8235–8241. w27x R.B. Painter, R.B. Young, Radiosensitivity in ataxia– telangiectasia: a new explanation, Proc. Natl. Acad. Sci. U. S. A. 77 Ž1980. 7315–7317. w28x R.B. Painter, Inhibition of mammalian cell DNA synthesis by ionizing radiation, Int. J. Radiat. Biol. 49 Ž1986. 771–781. w29x A. Lallev, B. Anachkova, G. Russev, Effect of ionizing radiation and topoisomerase II inhibitors on DNA synthesis in mammalian cells, Eur. J. Biochem. 216 Ž1993. 177–181. w30x J.A. D’Anna, J.G. Valdez, R.C. Habbersett, H.A. Crissman, Association of G1rS-phase and late S-phase checkpoints with regulation of cyclin-dependent kinases in Chinese hamster ovary cells, Radiat. Res. 148 Ž197. 260–271. w31x J.M. Larner, H. Lee, J.L.S. Hamlin, S phase damage sensing checkpoints in mammalian cells, Cancer Surv. 29 Ž1997. 25–45. w32x J.S. Bedford, Sublethal damage, potentially lethal damage, and chromosomal aberrations in mammalian cells exposed to ionizing radiation, Int. J. Radiat. Oncol., Biol., Phys. 21 Ž1991. 37–42. w33x T.J. McMillan, J.K. Peacock, Molecular determinants of radiosensitivity in mammalian cells, Int. J. Radiat. Biol. 65 Ž1994. 49–55. w34x D.T. Goodhead, Initial events in the cellular effects of ionizing radiations: clustered damage in DNA, Int. J. Radiat. Biol. 65 Ž1994. 7–17.