Mutagenesis, Vol. 15, No. 1, 25-31,
January 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Molecular analysis of 5-azacytidine-induced variants in mammalian cells
1 Cancer Genetics Group, National Institutes of Health, Research Triangle Park, NC 27709, USA and 2 Orszagos Kemiai Biztonsagi Intezet Gyali ut 2–6, Budapest 1966, Hungary
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
5-Azacytidine induces 6-thioguanine resistance in AS52 cells. To characterize these resistant clones, we isolated 148 of them from 50 independently treated flasks. Less than nine (6%) of the 148 variants were spontaneous. PCR amplification of the DNA primers flanking the gpt gene produced no product in 15 clones (10%). Of the 133 remaining clones, 52 showed sequence alterations in the gpt structural gene. Of these 52, 34 (65%) were GC
CG transversions. Only seven were located in CpG sequences. Thus, methyltransferase complexes are not major contributors to 5-azacytidine-induced point mutations in AS52 cells. The remaining 81 clones had no sequence alterations within the coding region of the gpt gene. Southern blot analysis of a sample of these variants (37/81) indicated that the 6-thioguanine-resistant phenotype was not due to local rearrangements or deletions (resolution 50 bp). Sequence analysis of the early promoter region of another sample of these variants (24/81) indicated that lesions in the promoter could not be responsible for the 6-thioguanine resistance observed. Thus, a majority of these variants were formed via a mechanism other than small genomic rearrangements, point mutations or deletions of the gpt structural gene or its promoter. Neither the mechanisms leading to these variants nor the biological and morphological consequences of these variants are known. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
5-Azacytidine is a cytidine analog in which a nitrogen replaces carbon in the 5 position of the pyrimidine ring. Its deoxy form is incorporated into DNA in place of cytidine during replication. This is a random process. When incorporated into CpG sequences, 5-azacytidine can inhibit methyltransferase activity by forming covalent complexes with this enzyme. This blocks the processivity of the enzyme and prevents normal post-replication methylation of DNA. 5-Aza-2'-deoxycytidine has been shown to be mutagenic and it has been postulated that this process is mediated by the formation of 5-aza-2'-deoxycytidine–methyltransferase complexes (Jackson Grusby et al., 1997
).
5-Azacytidine was originally developed as a chemotherapeutic agent for the treatment of human myelogenous leukemia (Von Hoff et al., 1976). More recently, the deoxy form, 5-aza-2'-deoxycytidine, has been shown to reverse epigenetic silencing of tumor suppressor genes (Jones, 1996). 5-Azacytidine was carcinogenic in mice and rats (NCI, 1978; Carr et al., 1984, 1988; Cavaliere et al., 1987). However, in a small study in rats, 5-aza-2'-deoxycytidine, which also causes hypomethylation, did not show carcinogenic activity (Carr et al., 1988). 5-Azacytidine transformed BHK and primary rat tracheal epithelial cell cells in culture (Bouck et al., 1984; Walker and Nettesheim, 1986). The authors of both studies suggested that transformation did not result from mutation, but this conclusion was a result of examining mutation at the ouabain or hprt loci. 5-Azacytidine was mutagenic in Escherichia coli, human lymphoblasts, Salmonella typhimurium and mouse lymphoma cells (Friedman, 1979; Podger, 1983; Zimmermann and Scheel, 1984; Call et al., 1986; Amacher and Turner, 1987; McGregor et al., 1989). Furthermore, 5-azacytidine induced micronuclei in mouse lymphoma cells (Stopper et al., 1992, 1995), apoptosis in thymocytes and HL-60 cells (Kizaki et al., 1992; Gorczyca et al., 1993) as well as a variety of chromosome aberrations including sister chromatid exchanges (Banerjee and Benedict, 1979), chromatid breaks (Karon and Benedict, 1972), achromatic gaps and chromosome fragmentation (Li et al., 1970; Benedict et al., 1977; Lavia et al., 1985; Walker et al., 1987; Stopper et al., 1993). 5-Azacytidine treatment leads to reversible cell cycle arrest (Spencer et al., 1996; Taylor,E.M. et al. 1996). DNA replication was a necessary prerequisite for the DNA damage, differentiation and altered gene expression observed in 5-azacytidine-treated cells (Stopper et al., 1993; Taylor,S.M., 1993). Repair of 5-azacytidine-mediated DNA damage required various pathways in yeast, including excision, mismatch and recombination repair (Hegde et al., 1996).
Jones and co-workers stimulated interest in the biological consequences of 5-azacytidine mediated by methylation mechanisms when they showed that this compound induced differentiation in C3H10T1/2 cells (Constantinides et al., 1977; Jones et al., 1982; Taylor,S.M. and Jones, 1982). Landolph and Jones (1982) suggested that mutation could not be the mechanism for its biological activity when they found that they could not correlate this biological activity of a series of 5-azacytidine analogs with their ability to induce resistance to ouabain and 8-azaguanine resistance in C3H10T1/2 and V79 cells, respectively. The ability of these compounds to methylate DNA, however, correlated with the biological activity (Jones and Taylor, 1980). We examined the mutagenic activities of these same azacytidine analogs using trifluorothymidine resistance in mouse lymphoma cells and found a correlation between induced trifluorothymidine resistance (McGregor et al., 1989) and each compound's reported ability to cause C3H10T1/2 cells to differentiate (Constantinides et al., 1977). This suggested that the ouabain and 8-azaguanine resistance used by Landolf and Jones may not have been appropriate models with which to test for the mutagenic activity of this compound and led us to examine the molecular basis of 5-azacytidine-induced mutation. The initial results of this investigation are reported here. Although a molecular analysis of 5-aza-2'-deoxycytidine-induced variants has been reported (Jackson Grusby et al., 1997), such an analysis has not been done on 5-azacytidine-induced variants.
We previously showed that 5-azacytidine induced trifluorothymidine resistance in L5178Y mouse lymphoma cells (McGregor et al., 1989). Because the mouse tk structural gene is large and not amenable to convenient sequence analysis of mutations, we decided to examine the molecular basis of variant formation in AS52 cells. The AS52 cell line is a genetically engineered hprt– CHO cell line that carries a single copy of the E.coli gpt gene (Tindall et al., 1984). The gpt gene expresses xanthine guanine phosphoribosyltransferase using the SV40 early promoter. The gpt gene is the functional equivalent of the mammalian hprt gene, except that xanthine-guanine phosphoribosyltransferase catalyzes the formation of xanthine 5'-monophosphate from xanthine while hypoxanthine-guanine phosphoribosyltransferase does not. The gpt coding sequence contains 459 bases including 111 cytidines, 41 of which are in CpG sequences (Figure 1). This gene is readily amplified by PCR for DNA sequence analysis. In addition, the AS52 cell line is known to detect both point and chromosomal mutations (Stankowski et al., 1986a,b). The data presented here show that 5-azacytidine induces point mutations in mammalian cells. It also reveals the presence of variants whose mechanism of formation and biological and morphological consequences are unknown.
|
represents a deletion. Mutations marked by * could be siblings. |
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Cell culture and mutant isolation
AS52 cells were routinely cultured in Ham's F12 selective medium (Gibco BRL) to eliminate any pre-existing 6-thioguanine (Sigma)-resistant mutants. Approximately 24 h prior to treatment with 5-azacytidine (Radian Corp.), 106 cells were plated in 25 cm2 tissue culture flasks in 10 ml of recovery medium consisting of Ham's F12.
Twenty-five separate cultures were treated with 3.0 µg/ml 5-azacytidine in 5 ml of Ham's F12. At approximately 20 h incubation after treatment, 106 cells from each culture were plated in fresh medium and maintained in log phase growth for 7 days. They were then selected for 6-thioguanine resistance by plating 106 cells in medium containing 10 µM 6-thioguanine. Surviving cells were incubated for a further 8 days before harvesting.
For mutant isolation, two plates were prepared for each culture treated with 5-azacytidine. Two colonies were isolated from each plate on day 15, for a total of 100 colonies. Cells from individual colonies were dispersed in 3 ml of selection medium in 12-well dishes and grown to confluency, then replated in tissue culture flasks and again grown to confluency. An aliquot of ~106 cells was pelleted for DNA extraction.
The entire experiment as described above was repeated to isolate an additional 100 mutant colonies.
DNA extraction
Extraction of genomic DNA from individual mutant colonies was performed using the ONCOR® Non-Organic DNA Extraction Kit.
PCR amplification and purification
Mutant DNA samples were amplified by PCR then analyzed by gel electrophoresis to determine the presence or absence of the gpt fragment in the mutant cell DNA. We performed two PCR reactions with each mutant clone. For the primary PCR reaction, we used primers p10/29 and p12/30 (Table I) flanking the gpt structural gene from –199 to +84 bases. We also amplified the dhfr gene as an internal PCR control. The amplified fragments were electrophoresed in 0.8% low melting point agarose gel. The analyzed PCR products normally showed three fragments: a 0.45 kb DHFR band, a 0.7 kb gpt band and a 1.1 kb rearrangement. This 1.1 kb fragment is a non-functional linked gpt rearrangement located downstream from the functional gpt gene. It is also flanked by the forward and reverse primers, 10/29 and 12/30 (Figure 2). A second set of primers flanking the dhfr gene was added as an internal control to ensure that the PCR reaction worked. A reaction showing no band corresponding to the gpt gene or the linked rearrangement that also showed no band corresponding to the dhfr gene was repeated until the band corresponding to the dhfr gene was observed.
|
|
For DNA sequence analysis, we excised the 0.7 kb gpt gene directly from the gel and performed a second PCR reaction using this fragment. In this PCR, the two primers were p5.1 and p12/30 (Table I
) flanking the gpt structural gene from –62 to +84 bases. After this secondary PCR, the amplified gpt gene was purified first with Strataclean Resin (Stratagene), then with a 100 kDa Ultrafree filter (Millipore). After the samples were resuspended in dH2O, DNA concentrations were measured with a spectofluorometer (Turner fluorometer) and dried using a Savant Speed-Vac concentrator. The samples were resuspended to 50 ng/ml in dH2O.
Sequence analysis
The Sanger dideoxy-mediated chain termination method was used to sequence the gpt structural gene. To sequence the entire structural gpt gene, two primers were used: p5.1 and p3, which are in the middle of the structural gene (Table I). The sequences with these primers overlap. The primers were end-labeled with [33P]ATP using T4 polynucleotide kinase (Biolabs). After the sequencing reaction, the samples were run in a 5.5% urea–bis-acrylamide vertical gel. Using p5.1, the first half of the gpt gene was sequenced. The rest of the gene was sequenced only if no mutation was found in the first half.
Southern blot analysis
To obtain a Southern blot we digested 15 µg of genomic DNA with BamHI and HindIII restriction enzymes, respectively. The digested DNA was purified using the methanol precipitation method and electrophoresed in a 1% agarose gel overnight. The following day the DNA was transferred to a Duralon-UV membrane (Stratagene) and immobilized with UV.
The membrane was hybridized with 50 ng of linearized pSV2gpt plasmid, labeled with [
-32P]dCTP using Stratagene's Prime-it Random Primer kit.
Promoter PCR amplification and purification
The DNA samples were PCR amplified. Primers chosen for the PCR flanked the gpt structural gene, the entire early promoter and most of the late promoter of SV40. The primers were primer p1 at the 5'-end and primer p12/30 at the 3'-end (Table I). We used Dynazyme II DNA polymerase (Finnzymes Oy) at 1 U/reaction. The amplified fragments were separated and visualized by electrophoresis at 100 V for 1 h through a 1% agarose gel containing 0.6 mg/ml ethidium bromide.
To purify the PCR products, we added a 1:1 vol of phenol-chloroform-isoamylalcohol (PCI) and centrifuged at 10 000 r.p.m. for 1 min. This was repeated using chloroform-isoamylalcohol (CI). Isoproponal (200 ml) was added and the mixture centrifuged at 10 000 r.p.m. for 10 min. We then washed the pellet with 400 ml 70% ethanol and centrifuged at 10 000 r.p.m. for 5 min. After drying in air, the pellet was suspended in 25 ml distilled water.
Sequence analysis of the promoter
The Sanger dideoxy-mediated chain termination method was used to sequence the promoter region. The primer chosen was FSTP (F-CAA CAT GTC CCA GGT GAC GAT GTA-T) fluorescein labeled at the 5'-end, which binds in the gpt structural gene to nt 36 of the antisense strand. For sequencing we used an ALF DNA Sequencer (Pharmacia) with the ThermoSequenase fluorescent labeled primer cycle sequencing kit (Amersham) according to the protocol provided.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Previously, we established that 3 µg/ml 5-azacytidine and an expression time of 7 days maximized the mutant fraction of 5-azacytidine-treated AS52 cells (Spencer et al., 1996
). Using these conditions, we treated 50 independent cell cultures with 5-azacytidine in two experiments and isolated 6-thioguanine-resistant AS52 clones for molecular analysis. We isolated four 6-thioguanine-resistant clones from each culture so that we would have at least 50 independent cultures. Of the isolated cultures, 148 survived. In these studies, we recorded the identity of the culture of all isolated clones so that we could assess clonal independence. Thus, if two clones from the same culture had the same lesion, we identified them as potential siblings and only one of them was considered for further analysis. The induced mutant fraction ranged from 415 to 750x10–6 and the average relative mutant fraction (the ratio of the mutant fraction of the treated culture to that of the untreated culture) was 15.9 for the two experiments. This indicates that for every 16 6-thioguanine-resistant colonies isolated, only one can be expected to be spontaneous. Consequently, >96% of the variants are 5-azacytidine-induced. Our strategy was to first identify those clones that have the gpt gene deleted and then to identify those that harbored intragenic mutations by sequencing the coding region of the remaining clones.
Analysis for chromosomal mutations
We used PCR analysis to identify those clones harboring deletions of the gpt gene. Using primers that flank the gpt gene, we observed two bands in wild-type AS52 cells. These cells contain, in addition to the functional gpt gene, a linked gpt rearrangement (Figure 2) (Tindall and Stankowski, 1989). This includes 79 bp of the 5'-region of the gene, a 0.7 kb insertion and 60 bp from the 3'-region of the gene (Z.Kelecsényi and K.R.Tindall, unpublished data). Primers that flank the gpt gene will also amplify the linked rearrangement (Table I). Thus, PCR amplification gave rise to a 0.7 kb fragment from the functional gpt gene and a 1.1 kb fragment from the non-functional rearrangement (Figure 3). We also included primers flanking the dhfr gene as an internal control. Consequently, electrophoresis of the PCR products from wild-type DNA produced three bands.
|
PCR amplification of the structural portion of the gene showed that 15 of the 148 6-thioguanine-resistant clones (10%) lacked a band at the site of the gpt gene. These 15 clones came from 12 different cultures, indicating that at least 12 of these were independent. Among these 12, 10 also lacked the 1.1 kb linked gpt rearrangement. The other two mutants lacked only the gpt gene. In each of the remaining 133 samples containing the gpt gene, the linked gpt rearrangement was also present.
Sequence analysis of the gpt structural gene
Sequence analysis of the remaining 133 mutant clones showed that 52 had point mutations (Figure 1 and Table II). Thirty-nine of the 52 were transversions: 34 GCCG, four GCTA, one ATCG but no ATTA mutations. Two of the 52 were transitions, GCAT. Ten of the 52 were small deletions, including eight 3 bp deletions and two 1 bp mutants, possibly siblings. Six of the eight deletion mutants had the same 3 bp sequence loss found in spontaneous mutants from this cell line. The last of the 52 mutants had a complex mutation where a TTT sequence replaced the 5 bp sequence GCTTA between 209 and 215.
|
Southern blot analysis
Of the 133 sequenced clones, 81 showed no alteration in the gpt structural gene. These 81 clones came from 37 separate cultures. We sampled these 81 clones by choosing no more than one clone from each of these cultures for the remainder of this work. This also ensured that no siblings were analyzed.
Deletions either within or of the gpt integration site could be responsible for some or all of the 6-thioguanine resistance of these 81 clones. We performed Southern blot analyses of an ~9 kb fragment around the gpt gene to determine if there was a rearrangement or deletion causing the gpt gene to be inactive. We examined 37 independent clones using linearized pSV2gpt plasmid as a probe. This plasmid was used to create AS52 cells from CHO cells. BamHI does not cut within the sequence that includes the SV40 promoter and the gpt structural gene (Figure 2). However, there are three BamHI restriction sites: one is 2.5 kb upstream from the SV40 promoter, a second 1.25 kb downstream from the structural gpt gene and a third 1.6 kb downstream from the linked rearrangement (Figure 2). A Southern blot gives rise to two bands, one at 4.2 and the other at 4.7 kb.
HindIII cuts upstream from the beginning of the SV40 sequence, at the 3'-end of the SV40 promoter region and at the 3'-end of the rearrangement (Figure 2). Restriction with this enzyme also gives rise to two bands. The 1.8 kb fragment contains the promoter region and sequences upstream; the 4.5 kb fragment contains the gpt gene and sequences downstream.
None of the 37 independent clones examined using Southern blot analysis showed changes in the restriction endonuclease pattern, as compared with the wild-type. This indicated that within the resolution of the gel (~50 bp), there were no deletions or genomic rearrangements in an ~9 kb fragment of the genomic DNA including the gpt integration site.
Thus far, we have not ruled out small deletions or point mutations within the promoter region. The gpt inserted sequence harbors both an early and a late promoter. In SV40 as in the AS52 cell line, the early and late promoters have different orientations. It is the SV40 early promoter that expresses the bacterial gene for XGPRT in mammalian cells (Tindall et al., 1984). For the last experiment, we focused our attention on this early promoter even though only a very low percentage showed mutations in this promoter region in previous work on the mutant spectrum of chemicals in AS52 cells (Stankowski et al., 1986b; Tindall et al., 1986; Tindall and Stankowski, 1989).
Sequence analysis of the promoter region
We sequenced the whole early and most of the late promoter region of 24 of the 37 independent clones. We were not able to sequence the entire late promoter because designing primers that included the entire late promoter was not possible since the sequence adjacent to the late promoter was unknown. However, it is only the early promoter that drives expression of the gpt gene. Our analysis showed that one of these had a 21 bp deletion which was located in the late promoter region, ~100 bp from the early promoter. It is not known whether this structural lesion is responsible for the 6-thioguanine resistance observed. However, since only one of the 24 resistant clones revealed a lesion in the late promoter, it is not important for the interpretation of these results. The remaining 23 were identical to each other and to promoter sequences derived from wild-type AS52 cells. We were able to find no point mutations or deletions in either the structural or promoter sequences of these 23 clones. Since these clones were picked randomly from the individual cultures, this implies that very few, if any, of the remaining 57 of the total of 81 cultures showing no structural changes would have shown point mutations or deletions of or within the early promoter region had they been examined.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Our data show that 5-azacytidine induces point mutations in mammalian cells. Fifty-two (35%) clones exhibited mutations in the gpt structural gene. Eleven of these were small (
3 bp) deletions or a complex mutation. In the remaining 41, the most frequent point mutation, was GCCG transversion, which made up 83% of the total point mutations found in the gpt structural gene. GCCG transversions have also been found in 5-azacytidine-treated bacteria and in a lacI transgene in the mouse (Levin and Ames, 1986; Watanabe et al., 1994; Jackson Grusby et al., 1997).
The distribution of the point mutations in the gpt structural gene did not suggest a potential hot spot. Of 34 GCCG transversions, 17 involve C in PyCPy, suggesting that there may be a slight bias for mutations at cytidines that are surrounded by pyrimidines. Of these 34 GCCG transversions, only seven occur at CpG sequences, which are the primary sites for methyltransferase binding in mammalian cells.
Jaenisch and co-workers studied the LacI transgene in the Big Blue mouse and found that 75% of the mutations in 5-aza-2'-deoxycytidine-treated mice occurred at CpG dinucleotides (Jackson Grusby et al., 1997). These workers speculated that G5-aza-CCG transversions at CpG sequences were mediated by methyltransferase–5-aza-2'-deoxycytidine complexes. The covalent adduct of this complex may be chemically unstable, resulting in ring opening leading to the transversion. They also proposed that this is the major mechanism for the formation of point mutations in their system. In our system, we find that only 20% of the point mutations (and only 5% of the total number of variants isolated) were located at CpG dinucleotides. This suggests that this mechanism, though it may be responsible for these mutations, could not be the major mechanism by which 5-azacytidine induced point mutations in AS52 cells.
Both mispairing and error-prone repair mechanisms have been invoked to explain 5-azacytidine-induced GCCG transversions in bacteria. 5-Azacytidine incorporates into DNA as though it were a cytidine, which may cause mispairing (Paces et al., 1968). Evidence of mispairing comes from mutation studies showing that this compound induced GCCG transversions in SOS repair-independent strains of Salmonella (Levin and Ames, 1986). Evidence for error-prone repair as a mechanism comes from work examining 5-azacytidine mutagenicity in various different strains of Salmonella and finding that the presence of plasmid pkM101 increased the sensitivity of these strains substantially (Schmuck et al., 1988). This plasmid contains the mucAB operon which enables the cell to perform SOS functions (Elledge et al., 1983) which are a prerequisite for error-prone repair (Doskocil et al., 1967; Witkin and Parisi, 1974; Ronen, 1980; Pietrzykowska et al., 1985).
Shifts in the nucleotide pool have also been known to induce mutations (Kunz et al., 1994) and it is possible that treatment with 5-azacytidine, which is structurally similar to cytidine, changes the composition of the nucleotide pool, altering the cell chemistry and resulting in GCCG transversions. In an earlier study, we found that cytidine treatment of L5178Y mouse lymphoma cells increased the mutation fraction over background (McGregor et al., 1989).
Methylcytosine has a propensity to undergo deamination to form thymidine (Coulondre et al., 1978; Wang et al., 1982). This has been proposed as a mechanism for GCAT transitions when the cytosine is methylated. It has been suggested that mutations produced by this mechanism may contribute significantly to human genetic disease (Cooper, 1983). However, it appears that 5-azacytidine does not promote this mechanism since only two of the mutants show a GCAT transition, the genotype that would be produced by deamination.
Some of the 15 deletion mutants are probably of spontaneous origin. Our measurement of a relative mutant fraction of 15.9 indicates that, in this population of 148 variant clones, approximately nine (6%) are of spontaneous origin. An analysis of spontaneous mutants in AS52 cells revealed that 45% are missing the gpt gene (Stankowski et al., 1986a,b; Tindall et al., 1989). Therefore, approximately four of the 15 mutants that showed these deletions could have arisen spontaneously. Thus only ~11 of the 148 6-thioguanine-resistant colonies could have been 5-azacytidine induced. We conclude that 6-thioguanine resistance in AS52 cells following 5-azacytidine treatment does not arise primarily as a result of deletion mutations.
5-Azacytidine has been reported to be clastogenic. An analysis of the reported data suggests that some of these observations occurred only at doses higher than the ones we used to generate our 6-thioguanine-resistant colonies. A National Toxicology Program study showed 5-azacytidine to increase the incidence of chromosomal aberrations marginally in CHO cells at 
10 µg/ml (E.Zeiger, personal communication). Fuciik and co-workers showed that 5-azacytidine is clastogenic in the root tips of Vicia faba at 25 µg/ml but they used only this one dose. 5-Azacytidine treatment of human TK-6 cells for 24 h up to a dose of 10 µg/ml induced no increase in the incidence of aberrant, polyploid or endoreduplicated cells and did not cause chromosome decondensation (Call et al., 1986). The dose–response curve for 6-thioguanine resistance in AS52 cells increases to a maximum value at ~3 µg/ml. This may explain why we do not observe many 6-thioguanine-resistant clones containing chromosomal aberrations. If 5-azacytidine produces chromosomal aberrations at these higher doses, it may imply that cells with these lesions do not survive to become viable mutants. However, treatment of L5178Y mouse cells and SHE cells with 0.25 µg/ml 5-azacytidine increased the number of micronuclei containing chromosomal fragments (Stopper et al., 1992, 1993), suggesting that lower doses can be clastogenic. It is not known whether these micronucleated cells survive to form viable mutants.
Although 67 (including the deletion mutants) of the clones manifested mutations in the coding region of the gene, 81 did not. We examined these for possible rearrangements or deletions in a region immediately surrounding the gpt insertion site. Our Southern blots (data not shown) were identical to those of the wild-type cells, suggesting that these mechanisms did not account for these 6-thioguanine resistance variants. Similarly, sequence analysis of the early promoter region did not suggest a mechanism to account for these variants. Thus, our data also show that 5-azacytidine may induce 6-thioguanine resistance in AS52 cells via a mechanism other than small genomic rearrangements, point mutations or deletions of the gpt structural gene.
|
Acknowledgments |
|---|
We wish to thank Professor Roger Adams of the University of Glasgow and Dr Daniella Carotti of the University of Rome for reading this manuscript and to Dr Kenneth Tindall at NIEHS for his help in this work. Z.K. was a fellow of the Fogarty International Center.
|
Notes |
|---|
* To whom correspondence should be addressed. Tel: +1 919 541 2150; Fax: +1 919 541 2242; Email: caspary{at}niehs.nih.gov
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Amacher,D. and Turner,G. (1987) The mutagenicity of 5-azacytidine and other inhibitors of replicative DNA synthesis in the L5178Y mouse lymphoma cell. Mutat. Res., 176, 123–131.[ISI][Medline]
Banerjee,A. and Benedict,W. (1979) Production of sister chromatid exchanges by various cancer chemotherapeutic agents. Cancer Res., 39, 797–799.
Benedict,W.F., Banerjee,A., Gardner,A. and Jones,P.A. (1977) Induction of morphological transformation in mouse C3H/10T1/2 clone 8 cells and chromosomal damage in hamster A (T1)C1-3 cells by cancer chemotherapeutic agents. Cancer Res., 37, 2202–2208.[ISI][Medline]
Bouck,N., Kokkinakis,D. and Ostrowsky,J. (1984) Induction of a step in carcinogenesis that is normally associated with mutagenesis by nonmutagenic concentrations of 5-azacytidine. Mol. Cell. Biol., 4, 1231–1237.
Call,K., Jensen,J., Liber,H. and Thilly,W. (1986) Studies of mutagenicity and clastogenicity of 5-azacytidine in human lymphoblasts and Salmonella typhimurium. Mutat. Res., 160, 249–257.[ISI][Medline]
Carr,B.I., Reilly,J.G., Smith,S.S., Winberg,C. and Riggs,A. (1984) The tumorigenicity of 5-azacytidine in the male Fischer rat. Carcinogenesis, 5, 1583–1590.
Carr,B.I., Rahbar,S., Asmeron,Y., Riggs,A. and Winberg,C.D. (1988) Carcinogenicity and haemoglobin synthesis induction by cytidine analogues. Br. J. Cancer, 57, 395–402.[ISI][Medline]
Cavaliere,A., Bufalari,A. and Vitali,R. (1987) 5-Azacytidine carcinogenesis in BALB/c mice. Cancer Lett., 37, 51–58.[ISI][Medline]
Constantinides,P.G., Jones,P.A. and Gevers,W. (1977) Functional striated muscle cells from non-myoblast precursors following 5-azacytidine treatment. Nature, 267, 364–366.[Medline]
Cooper,D. (1983) Eukaryotic DNA methylation. Hum. Genet., 64, 315–333.[ISI][Medline]
Coulondre,C., Miller,J.H., Farabaugh,P.J. and Gilbert,W. (1978) Molecular basis of base substitution hotspots in Escherichia coli. Nature, 274, 775–780.[Medline]
Doskocil,J., Paces,V. and Sorm,F. (1967) Inhibition of protein synthesis by 5-azacytidine in Escherichia coli. Biochim. Biophys. Acta, 145, 771–779.[Medline]
Elledge,S., Perry,K., Krueger,J.M.,B. and Walker,G. (1983) Cellular components required for mutagenesis. In Friedberg,E. and Bridges,B. (eds), Cellular Responses to DNA Damage. Wiley Liss, New York, NY, pp. 353–359.
Friedman,S. (1979) The effect of 5-azacytidine on E.coli DNA methylase. Biochem. Biophys. Res. Commun., 89, 1328–1333.[ISI][Medline]
Gorczyca,W., Gong,J., Ardelt,B., Traganos,F. and Darzynkiewicz,Z. (1993) The cell cycle related differences in susceptibility of HL-60 cells to apoptosis induced by various antitumor agents. Cancer Res., 53, 3186–3192.
Hegde,V., McFarlane,R.J., Taylor,E.M. and Price,C. (1996) The genetics of the repair of 5-azacytidine-mediated DNA damage in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet., 251, 483–492.[ISI][Medline]
Jackson Grusby,L., Laird,P.W., Magge,S.N., Moeller,B.J. and Jaenisch,R. (1997) Mutagenicity of 5-aza-2'-deoxycytidine is mediated by the mammalian DNA methyltransferase. Proc. Natl Acad. Sci. USA, 94, 4681–4685.
Jones,P.A. (1996) DNA methylation errors and cancer. Cancer Res., 56, 2463–2467.
Jones,P.A. and Taylor,S.M. (1980) Cellular differentiation, cytidine analogs and DNA methylation. Cell, 20, 85–93.[ISI][Medline]
Jones,P., Taylor,S., Mohandas,T. and Shapiro,L. (1982) Cell cycle-specific reactivation of an inactive X-chromosome locus by 5-azadeoxycytidine. Proc. Natl Acad. Sci. USA, 79, 1215–1219.
Karon,M. and Benedict,W.F. (1972) Chromatid breakage: differential effect of inhibitors and synthesis during G2 phase. Science, 178, 62.
Kizaki,H., Ohnishi,Y., Azuma,Y., Mizuno,Y. and Ohsaka,F. (1992) 1-ß-D-Arabinosylcytosine and 5-azacytidine induce internucleosomal DNA fragmentation and cell death in thymocytes. Immunopharmacology, 24, 219–227.[ISI][Medline]
Kunz,B.A., Kohalmi,S.E., Kunkel,T.A., Mathews,C.K., McIntosh,E.M. and Reidy,J.A. (1994) International Commission for Protection Against Environmental Mutagens and Carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability. Mutat. Res., 318, 1–64.[ISI][Medline]
Landolph,J.R. and Jones,P.A. (1982) Mutagenicity of 5-azacytidine and related nucleosides in C3H10T1/2 Clone 8 and V79 cells. Cancer Res., 42, 817–823.
Lavia,P., Ferraro,M., Micheli,A. and Olivieri,G. (1985) Effect of 5-azacytidine (5-aza C) on the induction of chromatid aberrations (CA) and sister-chromatid exchanges (SCE). Mutat. Res., 149, 463–467.[ISI][Medline]
Levin,D.E. and Ames,B.N. (1986) Classifying mutagens as to their specificity in causing the six possible transitions and transversions: a simple analysis using the Salmonella mutagenicity assay. Environ. Mutagen., 8, 9–28.[ISI][Medline]
Li,L.H., Olin,E.J., Fraser,T.J. and Bhuyan,B.K. (1970) Phase specificity of 5-azacytidine against mammalian cells in tissue culture. Cancer Res., 30, 2770–2775.
McGregor,D.B., Brown,A.G., Cattanach,P., Shepherd,W., Riach,C., Daston, D.S. and Caspary,W.J. (1989) TFT and 6TG resistance of mouse lymphoma cells to analogs of azacytidine. Carcinogenesis, 10, 2003–2008.
NCI (1978) Bioassay of 5-Azacytidine for Possible Carcinogenicity (CAS No. 320-67-2), National Cancer Institute Carcinogenesis Technical Report Series no. 42, DHHS (NIH) Publication no. 78.842. National Institutes of Health, Bethesda, MD.
Paces,V., Doskocil,J. and Sorm,F. (1968) Incorporation of 5-azacytidine into nucleic acids of Escherichia coli. Biochim. Biophys. Acta, 161, 352–360.[Medline]
Pietrzykowska,I., Krych,M. and Shugar,D. (1985) Involvement of DNA lesions and SOS functions in 5-bromouracil-induced mutagenesis. Mutat. Res., 149, 287–296.[ISI][Medline]
Podger,D.M. (1983) Mutagenicity of 5-azacytidine in Salmonella typhimurium. Mutat. Res., 121, 1–6.[ISI][Medline]
Ronen,A. (1980) 2-Aminopurine. Mutat. Res., 75, 1–47.[ISI][Medline]
Schmuck,G., Lieb,G., Wild,D., Schiffmann,D. and Henschler,D. (1988) Characterization of a in vitro micronucleus assay with syrian hamster embryo fibroblasts. Mutat. Res., 203, 397–404.[ISI][Medline]
Spencer,D.L., Caspary,W.J., Hines,K. and Tindall,K.R. (1996) 5-Azacytidine induced 6-thioguanine-resistance at the gpt locus in AS52 cells: cellular response. Environ. Mol. Mutagen., 28, 100–106.[ISI][Medline]
Stankowski,L.F.Jr, Leon F. and Hsie,A.W. (1986a) Quantitative and moleclar analyses of radiation-induced mutation in AS52 cells. Radiat. Res., 105, 37–48.[ISI][Medline]
Stankowski,L.F.Jr, Tindall,K.R. and Hsie,A.W. (1986b) Quantitative and molecular analyses of ethyl methanesulfonate- and ICR 191-induced mutation in AS52 cells. Mutat. Res., 160, 133–147.[ISI][Medline]
Stopper,H., Pechan,R. and Schiffmann,D. (1992) 5-Azacytidine induces micronuclei in and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA synthesis. Mutat. Res., 283, 21–28.[ISI][Medline]
Stopper,H., Körber,C., Schiffmann,D. and Caspary,W.J. (1993) Cell cycle dependent micronucleus formation and mitotic disturbances in mammalian cells treated with 5-azacytidine. Mutat. Res., 300, 165–177.[ISI][Medline]
Stopper,H., Körber,C., Gibis,P., Spencer,D.L. and Caspary,W.J. (1995) Micronuclei induced by modulators of methylation: analogs of 5-azacytidine. Carcinogenesis, 16, 1647–1650.
Taylor,E.M., McFarlane,R.J. and Price,C. (1996) 5-Azacytidine treatment of the fission yeast leads to cytotoxicity and cell cycle arrest. Mol. Gen. Genet., 253, 128–137.[ISI][Medline]
Taylor,S.M. (1993) 5-Aza-2'-deoxycytidine: cell differentiation and DNA methylation. Leukemia, 1, 3–8.
Taylor,S.M. and Jones,P.A. (1982) Mechanism of action of eukaryotic DNA and methyltransferase. J. Mol. Biol., 162, 679–692.[ISI][Medline]
Tindall,K.R. and Stankowski,L.F.Jr (1989) Molecular analysis of spontaneous mutations at the gpt locus in Chinese hamster ovary (AS52) cells. Mutat. Res., 220, 241–253.[ISI][Medline]
Tindall,K.R., Stankowski,L.F.Jr, Machanoff,R. and Hsie,A.W. (1984) Detection of deletion mutations in pSV2gpt-transformed cells. Mol. Cell. Biol., 4, 1411–1415.
Tindall,K.R., Stankowski,L.F.Jr, Machanoff,R. and Hsie,A.,W. (1986) Analyses of mutation in pSV2gpt-transformed CHO cells. Mutat. Res., 160, 121–131.[ISI][Medline]
Von Hoff,D., Slavik,M. and Muggia,F. (1976) 5-Azacytidine: a new anticancer drug with effectiveness in acute myelogenous leukemia. Ann. Intern. Med., 85, 237–245.
Walker,C. and Nettesheim,P. (1986) In vitro transformation of primary rat tracheal epithelial cells by 5-azacytidine. Cancer Res., 46, 6433–6437.[ISI][Medline]
Walker,C., Matthews,A. and Shay,J. (1987) Suppression of tumorigenicity mediated by 5-azacytidine and associated with increased chromosome number. J. Natl Cancer Inst., 78, 695–700.
Wang,R., Kuo,K., Gehrke,C., Huang,L. and Ehrlich,M. (1982) Heat and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA. Biochim. Biophys. Acta, 697, 371–377.[Medline]
Watanabe,M., Nohmi,T. and Ohta,T. (1994) Effects of the umuDC, mucAB and samAB operons on the mutational specificity of chemical mutagenesis in Escherichia coli: II. Base substitution mutagenesis. Mutat. Res., 314, 39–49.[ISI][Medline]
Witkin,E.M. and Parisi,E.C. (1974) Bromouracil mutagenesis: mispairing or misrepair? Mutat. Res., 25, 407–409.[ISI][Medline]
Zimmermann,F.K. and Scheel,I. (1984) Genetic effects of 5-azacytidine in Saccharomyces cerevisiae. Mutat. Res., 139, 21–24.[ISI][Medline]
Received on April 13, 1999; accepted on August 19, 1999.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  B. H. Lee, S. Yegnasubramanian, X. Lin, and W. G. Nelson Procainamide Is a Specific Inhibitor of DNA Methyltransferase 1 J. Biol. Chem., December 9, 2005; 280(49): 40749 - 40756. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||