Mutagenesis Advance Access originally published online on January 31, 2007
Mutagenesis 2007 22(3):177-181; doi:10.1093/mutage/gel069
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Mechanism of in vivo sister-chromatid exchange induction by 5-azacytidine
Instituto Nacional de Investigaciones Nucleares, AP 18-1027 México, D.F., México
The aim of the present study was to explore the in vivo mechanism of sister-chromatid exchange (SCE) induction by 5-azacytidine (5-azaC) in murine bone marrow cells. Experiments were performed to examine SCE induction in response to different doses of 5-azaC as well as several exposures. Additionally, we examined the persistence of SCE induction and the effect of bromodeoxiuridine (BrdU) incorporation. Sister-chromatid differentiation was obtained by injecting mice intraperitoneally with BrdU absorbed to activated charcoal. Before BrdU injection, different doses of 5-azaC were administered intraperitoneally either singly or in multiples. Colchicine in an aqueous solution was administered subcutaneously 22 h after BrdU injection. Two hours later, animals were sacrificed by cervical dislocation and both femurs were dissected. Bone marrow cells were processed to obtain chromosome preparations, which were stained by the fluorescence plus Giemsa method. Results indicate that 5-azaC caused SCE, albeit to a limited extent. In order to discern whether the limitation was due to cytotoxicity or to partial 5-azaC incorporation, we administered multiple sub-toxic doses of 5-azaC. This treatment increased 5-azaC incorporation and reduced cytotoxicity, but did not raise SCE frequency, indicating that the limitation was not due to either of the two factors mentioned above. SCE frequency induced by 5-azaC persisted for at least eight cell divisions, confirming that this agent had caused inhibition of DNA methyltransferase and subsequently the reduction of DNA re-methylation, which in turn induced the expression of a number of SCE-prone sites. Finally, SCE induction in response to 5-azaC was completely dependent on BrdU incorporation. The data allow us to conclude that 5-azaC causes SCE to a limited extent; limited SCE induction was not due to the direct effect of incorporation or cytotoxicity of 5-azaC, but rather the generation of a number of SCE-prone sites, the expression of which persists for at least eight cell divisions and is dependent on BrdU incorporation.
 |
Introduction |
|---|
 Top Introduction Materials and methodsResultsDiscussionReferences |
|---|
Sister-chromatid exchange (SCE) represents DNA double-strand exchange between chromatids of the same chromosome. According the more supported hypothesis, SCE induction represents recombination repair (1
) of DNA lesions that persist before duplication (2,3). However, a paradox exists due to evidence obtained from a three-way differential staining protocol, that agents could cause increased SCE frequencies that transcend cell division (4–7), although they do not seem to be caused at the same locus (8). The persistence of lesions involved in SCE for several cell divisions implies that lesions are not eliminated and SCE represents a mechanism to tolerate them. Evidence suggests that gamma rays induce a statistically significant increase in SCE frequency up to six cell divisions after exposure (9).
There is also evidence that DNA de-methylation causes a persistent and constant increase in SCE frequencies for more than eight cell divisions (10). This was interpreted as a result of an epigenetic alteration in DNA represented by de-methylation. A hypothesis that unifies the persistence of SCE induction caused by de-methylation and by alkylation of nucleophilic sites in DNA bases is that the latter is able to cause cytocine de-methylation in DNA by extensive repair of alkylated sites. In support of this possibility, a de-methylating effect of alkylating agents has been previously reported (11).
The inhibitory effect of 5-azacytidine (5-azaC) on DNA re-methylation is well established (12), as are the consequences of DNA de-methylation on several cell processes, such as mismatch repair (13), transduction control and genome stability (14). However, the mechanism that relates de-methylation with SCE induction is not clearly understood (15). Understanding this phenomenon is important because it would establish a link between DNA de-methylation, an epigenetic effect, and the persistent occurrence of homologous recombination.
Evidence with respect to the effect of 5-azaC on SCE induction in vitro include the following: (i) 5-azaC is able to induce significant increase in SCE (16), (ii) SCE frequency induced by this agent remains high and constant for several cell divisions (10) and (iii) SCE induction by 5-azaC showed a synergistic effect with various mutagens (17).
The aims of the present study were to determine the in vivo dose–response curve of SCE induction by 5-azaC in murine bone marrow cells, the persistence of SCE frequency induced after exposure to 5-azaC, the effect of several exposures to 5-azaC during the same cell division and the role of bromodeoxiuridine (BrdU) incorporation on SCE elicited by 5-azaC. All this was carried out in order to approach the mechanism of SCE induction by 5-azaC in the same biological system.
|
Materials and methods |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Animals
BALB/c male mice (2- to 3-month old) weighing 30 g were used in all experiments. Animals were bred and maintained in plastic cages with sawdust bedding under controlled conditions of temperature (22°C) and dark–light periods (light 7 am–7 pm). They were fed Purina chow for small rodents and given water ad libitum. Animals were treated and housed in accordance with the Guide for the Care and Use of Laboratory Animals (18
).
Chemicals
BrdU, 5-azaC and colchicine were obtained from Sigma-Aldrich (Quimica. México) Chemical Co.
Protocol
Anesthetized mice were intraperitoneally injected with a BrdU suspension absorbed to activated charcoal (19,20) of 250–300 mesh. BrdU dosage was 1.5 mg/g of body weight for most experiments. However, in some experiments the BrdU dose was an end point and, in such case, it varied in the range from 0.75 to 1.9 mg/g of body weight. 5-azaC was intraperitoneally administered in saline solution (0.9% NaCl) at the dosage given in the Results section. Treatment was applied 30 min before BrdU administration. Exceptions include experiments designed to examine the persistence of SCE induction, in which 5-azaC was administered 30 min or 24, 48 or 72 h before BrdU administration, as well as multiple 5-azaC treatment experiments, in which three or four doses of 5-azaC were injected every 3 h before BrdU administration. Colchicine was administered subcutaneously in aqueous solution at a dose of 3.75 mg/kg of body weight 22 h after BrdU injection. Two hours later, animals were sacrificed by cervical dislocation and both femurs were dissected to obtain bone marrow; bone marrow was obtained by injecting a phosphate-buffered saline solution into one end of the femur. The resultant cell suspension was centrifuged at 1500 r.p.m., re-suspended in 0.075 M KCl and incubated at 37°C for 15 min. It was then centrifuged again, re-suspended and fixed with three changes of methanol : acetic acid (3 : 1). Finally, the cell suspension was dropped onto clean, chilled slides.
Staining
Slides were dried for at least 24 h and stained using the fluorescence plus Giemsa method (21) slightly modified (22).
Analysis
SCE frequency was scored in 30 cells per mouse. The average generation time (AGT) was established according to a previously reported method (23). The mitotic index (MI) was determined as the number of metaphases per 1000 cells.
Statistical method
The statistical significance among groups was determined by the Student's t-test, using the Excel program for PC.
|
Results |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Dose response and cytotoxicity
Table I shows SCE induction data in murine bone marrow cells using different doses of 5-azaC. SCE frequency increased significantly with all doses. Doses of 20 and 40 µmoles/kg increased SCE frequency proportionally up to 2.0. A dose of 60 µmoles/kg, however, did not seem to cause any additional increase. The induction of SCE by 80 µmoles/kg was impossible to score because the number of cells with differential staining of sister chromatids was very low.
|
As showing in Table II, doses higher than 40 µmoles/kg, which caused maximal SCE induction, produced a cytotoxic effect in terms of both an increase in AGT and a reduction in MI. These doses produced an increase in AGT of 2.5 h or higher and a reduction in MI of about 40 %.
|
Multiple doses
As previously mentioned, high doses of 5-azaC proved to be cytotoxic, thereby limiting our ability to establish a dose–response relationship between 5-azaC dose and SCE induction. In order to explore whether higher 5-azaC incorporation increased SCE induction and reduced cellular toxicity, we exposed mice to several low doses spaced 3 h apart. The animals tolerated three doses of 40 µmoles/kg 5-azaC up to a total dose of 120 µmoles/kg, and although cytotoxicity was important, as can be seen in Table III, there were enough differentially stained cells for SCE scoring. Since four doses proved to be very cytotoxic, analysis of SCE was not possible. Results indicate that three doses did not cause an increase in SCE induction greater than that caused by a single acute dose of 40 µmoles/kg. This demonstrates that SCE induction was dependent either on dose or on DNA incorporation, but that there was a limit above which no additional increase was observed.
|
Persistent SCE induction
Table IV shows SCE induction by 5-azaC in successive divisions after exposure. Results reveal that SCE frequency increased at all time periods and was statistically significant with respect to untreated controls. The increase was almost identical for all periods determined. This implies that SCE remained high for a period of time equivalent to eight cell divisions, which was expressed in cell populations as observed in the curves of cumulative cell frequency versus SCE frequency shown in Figure 1.
|
|
Figure 2 shows the results of 5-azaC exposure on the MI and AGT several divisions after treatment. These parameters were affected by 5-azaC treatment in an inversely proportional manner, which means that AGT increased as MI decreased and vice versa. The complete concordance observed for this behaviour indicates that both events were caused by the same process.
|
Role of BrdU on SCE induction by 5-azaC
Table V presents the effect of BrdU dosage on SCE induction by 30 µmoles of 5-azaC. SCE induction only increased slightly, up to 0.5 SCEs per cell, when we administered a BrdU dose of 0.75 mg/g of body weight. This increase did not prove to be statistically significant with respect to control, which implies that 5-azaC did not induce SCE per se. However, a BrdU dose of 1.1 mg/g of body weight caused a clear increase of 1.6 SCEs per cell using the same dose of 5-azaC. Subsequent increases in BrdU dose resulted in SCE increases. Figure 3A shows the dose–response curves of BrdU induction of SCE in the presence or absence of 5-azaC. Slopes of curves from 5-azaC-treated and untreated control groups suggest that these curves would converge at lower 5-azaC doses; at the point of convergence, the SCEs should be induced only by BrdU. This result indicates that SCE induction in response to 5-azaC was completely dependent on BrdU incorporation. Figure 3B shows the curve of SCE increase caused by different doses of BrdU in cells treated with 5-azaC. The curve fit with a Bolzman sygmoidal curve (r = 1.0) having a plateau around 2.7 SCEs per cell indicates that there was a limited number of SCE-prone sites. This curve supplies evidence that SCE induction by 5-azaC was completely dependent on BrdU incorporation.
|
|
|
Discussion |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Our results indicate that 5-azaC also caused SCE in vivo, but with a lower frequency than that obtained in vitro (10
). Importantly, the induction of SCE in our in vivo study showed a clear limit. This could be explained by sensitization that resulted from the level of BrdU incorporation, given that basal SCE frequencies were substantially higher in previous in vitro studies (10,24). Use of a single acute dose and the proven increased cellular toxicity by higher doses of 5-azaC would suggest that limited SCE induction by an acute dose of 5-azaC is due to limited DNA incorporation or that the cytotoxic effect of higher 5-azaC doses did not allow a dose-dependent SCE increase. However, our results with multiple low doses of 5-azaC, causing a condition of reduced toxicity that allowed higher DNA incorporation, did not result in SCE increase. This suggests that these factors do not cause limited SCE induction but, possibly, the occurrence of a restricted number of prone SCE-occurring sites.
Based on previous evidence that 5-azaC inhibits methylation by sequestering molecules of DNA methylase (25), results of the present study could be explained by reduction of DNA re-methylation caused by 5-azaC, which results in the expression of limited SCE-prone sites.
Our results concerning the dependence of SCE induced by 5-azaC on BrdU incorporation, which were not previously reported, denote that both de-methylation and BrdU incorporation concurred in these SCE-prone sites.
The persistent SCE increase caused by 5-azaC for several divisions, which agrees with previously reported data (10), indicates that the epigenetic modification caused by de-methylation caused the appearance of SCE-eliciting sites in DNA which are revealed by BrdU. These sites are conceptually equivalent to fragile sites as evidenced by their small number and requirement for exposure to an agent to be manifested. In this context, our results are relevant to early observations that common fragile sites have been observed as hot spots not only for chromosomal lesions, such as gaps, but also for SCE formation (26). A remarkably high frequency of SCE was observed at gaps on common fragile sites after treatment with 5-azaC and BrdU (27). SCE has also been associated with fragile sites after treatment with aphidicolin (27), 4', 6-diamidino-2-phenylindole (28) and Mitomycin C (29).
Both DNA de-methylation and BrdU incorporation relax chromatin structure (30,31); the question is whether this determines susceptibility to the induction of lesions that could produce SCE or gaps arising from incomplete SCE. An additional explanation is that such a relaxation makes DNA more susceptible to damage induced by endogenous and exogenous mutagens. In fact, this hypothesis is also supported by permanent susceptibility or epigenetic susceptibility to mutagens induced by 5-azaC (10,17). Likewise, the scientific literature supports the sensitization to exogenous mutagens caused by both 5-azaC (17) and BrdU (2,3).
The hypothesis that SCE-prone sites caused by DNA de-methylation and subsequent BrdU incorporation are equivalent to fragile sites must be further explored.
Although the synergy of cytotoxic effects of BrdU and 5-azaC is not related with the aim of the present study, we consider it pertinent to comment on the subject. Our results indicate (Table V) that BrdU caused a slightly and not statistically significant cytotoxic effect, this determined as an increase in the AGT and as a reduction in MI; however, the pretreatment with 5-azaC caused an important and significant effect in both parameters. The importance of this resides in the possibility of using 5-azaC followed by BrdU as an anti-neoplastic treatment.
Results of the present in vivo study allow us to conclude that 5-azaC cause SCE to a limited extent and that limited SCE induction was not produced by the incorporation or cytotoxicity of 5-azaC, but rather by the epigenetic production of a number of SCE-prone sites, whose expression persists for at least eight cell divisions and is dependent on BrdU incorporation.
|
Acknowledgments |
|---|
This study was supported by the ‘Consejo Nacional de Ciencia y Tecnología' (National Council of Science and Technology) Project 33167-N. We wish to thank Angel Reyes, Perfecto Aguilar and Felipe Beltrán for their excellent technical assistance.
|
Notes |
|---|
* To whom correspondence should be addressed. Tel: +5329-7200; Fax: +525553297387 Email: pmr{at}nuclear.inin.mx
|
References |
|---|
|
TopIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
1. Matsuoka A, Lundin C, Johansson F, Sahlin M, Fukuhara K, Sjoberg BM, Jenssen D, Onfelt A. Correlation of sister chromatid exchange formation through homologous recombination with ribonucleotide reductase inhibition. Mutat. Res. (2004) 547:101–107.[Web of Science][Medline]
2. González-Beltrán F, Morales-Ramírez P. In vivo repair during G1 of DNA lesions eliciting sister chromatid exchanges induced by methylnitrosourea or ethylnitrosourea in BrdU substituted or unsubstituted DNA in murine salivary gland cells. Mutat. Res. (1999) 425:239–247.[Web of Science][Medline]
3. González-Beltrán F, Morales-Ramírez P. Repairability during G1 of lesions eliciting sister-chromatid exchanges induced by methylmethane sulfonate or ethylmethanesulfonate in BrdU-substituted and unsubstituted DNA strands. Mutagenesis (2003) 18:13–17.
4. Morales-Ramírez P, Vallarino-Kelly T, Rodríguez-Reyes R. Occurrence in vivo of sister chromatid exchanges at the same locus in successive cell divisions caused by nonrepairable lesions induced by gamma rays. Environ. Mol. Mutagen. (1988) 11:183–193.[Web of Science][Medline]
5. Morales-Ramírez P, Rodríguez-Reyes R, Vallarino-Kelly T. Fate of DNA lesions that elicit sister-chromatid exchanges. Mutat. Res. (1990) 232:77–88.[Web of Science][Medline]
6. Morales-Ramírez P, Rodríguez-Reyes R, Vallarino-Kelly T. In vivo fate of MMS-induced DNA lesions that elicit SCE. Mutat. Res. (1992) 272:215–221.[CrossRef][Web of Science][Medline]
7. Morales-Ramírez P, Rodríguez-Reyes R, Vallarino-Kelly T. Fate of lesions that elicit sister chromatid exchanges (FLE-SCE) produced in DNA by alkylating agents in vivo. Mutat. Res. (1995) 344:13–26.[CrossRef][Web of Science][Medline]
8. Rodríguez-Reyes R, Morales-Ramírez P. Sister-chromatid exchange induction and the course of DNA duplication, two mechanisms of SCE induction by ENU, and the role of BrdU. Mutagenesis (2003) 18:65–72.
9. Morales-Ramírez P, Vallarino-Kelly T, Rodríguez-Reyes R. In vivo persistence of sister chromatid exchange (SCE) induced by gamma rays in mouse bone marrow cells. Environ. Mutagen. (1984) 6:529–537.[Web of Science][Medline]
10. Perticone P, Paliti F, Cozzi R, D'Erme M, Bona R. Persistence of azacytidine-induced SCEs and genomic methylation in CHO cells in vitro. Mutat. Res. (1990) 245:211–215.[CrossRef][Web of Science][Medline]
11. Kastan MB, Gowans BJ, Lieberman MW. Methylation of deoxycytidine incorporated by excision-repair synthesis of DNA. Cell (1982) 30:509–516.[CrossRef][Web of Science][Medline]
12. Jones PA, Taylor SM. Cellular differentiation, cytidine analogs and DNA methylation. Cell (1980) 20:85–93.[CrossRef][Web of Science][Medline]
13. Hare JT, Taylor JH. One role for DNA methylation in vertebrate cells is strand discrimination in mismatch repair. Proc. Natl. Acad. Sci. USA (1985) 82:7350–7354.
14. Kress C, Thomassin H, Grange T. Local DNA demethylation in vertebrates: how could it be performed and targeted? FEBS Lett. (2001) 494:135–140.[CrossRef][Web of Science][Medline]
15. Albanesi T, Polani S, Cozzi R, Perticone P. DNA strand methylation and sister chromatid exchanges in mammalian cells in vitro. Mutat. Res. (1999) 429:239–248.[Web of Science][Medline]
16. Perticone P, Cozzi R, Gustavino B. Sister chromatid exchanges induced by DNA demethylating agents persist through several cell cycles in mammalian cells. Carcinogenesis (1987) 8:1059–1063.
17. Perticone P, Gensabella G, Cozzi R. Damage proneness induced by genomic DNA demethylation in mammalian cells cultivated in vitro. Mutagenesis (1997) 12:259–264.
18. Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council. Guide for the Care and Use of Laboratory Animals (1996) Washington, DC: National Academies Press. 140.
19. Morales-Ramírez P. Analysis in vivo of sister chromatid exchange in mouse bone-marrow and salivary-gland cells. Mutat. Res. (1980) 74:61–69.[Web of Science][Medline]
20. Morales-Ramírez P, Vallarino-Kelly T, Rodríguez-Reyes R. Detection of SCE in rodent cells using the activated charcoal bromodeoxyuridine system. Basic Life Sci. (1984) 29B:599–611.[Medline]
21. Perry P, Wolff S. New Giemsa method for the differential staining of sister chromatid. Nature (1974) 251:156–158.[CrossRef][Medline]
22. Goto K, Akematsu T, Shimazu H, Sugiyama T. Simple differential Giemsa staining of sister chromatid after treatment with photosensitive dyes and exposure to light and the mechanism of staining. Chromosoma (1975) 53:223–230.[CrossRef][Web of Science][Medline]
23. Ivett JL, Tice RR. Average generation time: a new method of analysis and quantitation of cellular proliferation kinetics. Environ. Mutagen. (1982) 4:358.
24. Shypley J, Sakai K, Tantravahi U, Fendrock B, Latt SA. Correspondence between effects of 5-azacytidine on SCE formation, cell cycling and DNA methylation in Chinese hamster cells. Mutat. Res. (1985) 150:333–345.[Web of Science][Medline]
25. Santi DV, Norment A, Garret CE. Covalent bond formation between a DNA-cytosine methyltransferase and DNA containing 5-azacytosine. Proc. Natl. Acad. Sci. USA (1984) 81:6993–6997.
26. Glower TW, Stein CK. Induction of sister chromatid exchanges at common fragile sites. Am. J. Hum. Genet. (1987) 41:882–890.[Web of Science][Medline]
27. Feichtinger W, Schmid M. Increased frequencies of sister chromatid exchanges at common fragile sites (1)(q42) and (19)(q13). Hum. Genet. (1989) 83:145–147.[CrossRef][Web of Science][Medline]
28. Gaddini L, Pellicia F, Limongi MZ, Rocchi A. Study of the relationships between common fragile sites, chromosome breakages and sister chromatid exchanges. Mutagenesis (1995) 10:257–260.
29. Wenger SL. Chemical induction of sister chromatid exchange at fragile sites. Cancer Genet. Cytogenet. (1995) 85:72–74.[CrossRef][Web of Science][Medline]
30. Satoh T, Yamamoto K, Miura KF, Sofuni T. Region-specific chromatin decondensation and micronucleus formation induced by 5-azacytidine in human TIG-7 cells. Cytogenet. Genome Res. (2004) 104:289–294.[CrossRef][Web of Science][Medline]
31. Zakharov AF, Egolina NA. Differential spiralization along mammalian mitotic chromosomes. Chromosoma (1972) 38:341–365.[CrossRef][Web of Science][Medline]
Received on October 19, 2006; revised on December 11, 2006; accepted on December 13, 2006.
CiteULike 
Connotea 
Del.icio.us What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||