Mutational-reporter transgenes rescued from mice lacking ... - Nature

Oncogene (2004) 23, 5931–5940

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Mutational-reporter transgenes rescued from mice lacking either Mgmt, or both Mgmt and Msh6 suggest that O6-alkylguanine-induced miscoding does not contribute to the spontaneous mutational spectrum Linda E Sandercock1, Melvin CH Kwok1, H Artee Luchman1, Sean C Mark1, Jennette L Giesbrecht1, Leona D Samson2 and Frank R Jirik*,1 1 Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada T2N 4N1; 2Biological Engineering Division and Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USA

O6-methylguanine methyltransferase, Mgmt, constitutes the first line of defense against O6-alkylguanine, which can result in G : C to A : T transitions upon DNA replication. Mgmt has been found in organisms as diverse as archaebacteria and mammals. This evolutionary conservation suggests that all organisms may be exposed to either endogenous or environmental alkylating agents. We thus hypothesized that tissues of Mgmt/ mice would exhibit elevated mutant frequencies. Employing the Big Bluet transgenic system, we evaluated lacI mutants rescued from liver and small intestinal DNA of young Mgmt/ mice. Interestingly, while there was a small difference between Mgmt/ mice and controls with respect to lacI mutant frequency, no differences attributable to Mgmt deficiency were apparent in the mutational spectra. Although mutations stemming from O6-guanine alkylations would be predicted to be cumulative, we found no evidence of an Mgmt-dependent alteration in mutation spectrum in DNA samples from 12 month-old mice. To optimize our ability to detect mutations resulting from O6alkylguanine-induced G : T mismatches, mice with combined deficiencies of Mgmt and the DNA mismatch repair molecule, Msh6, were analysed. In spite of this strategy, we observed no significant differences between Mgmt/ Msh6/ and Msh6/ mouse lacI mutations, except for a trend towards a greater percentage (of total transitions) of G : C to A : T changes in Mgmt/Msh6/ livers. Therefore, despite the striking evolutionary conservation of Mgmt, deficiency of this gene did not significantly impact the spontaneous lacI mutational spectrum in vivo. Oncogene (2004) 23, 5931–5940. doi:10.1038/sj.onc.1207791 Published online 21 June 2004 Keywords: Mgmt; Msh6; lacI; DNA mismatch repair; transgenic shuttle-phage; O6-alkylguanine; liver; small intestine; Big Bluet

*Correspondence: FR Jirik; E-mail: [email protected] Received 4 February 2004; revised 2 April 2004; accepted 2 April 2004; published online 21 June 2004

Introduction O6-methylguanine-DNA methyltransferase (Mgmt) (also referred to as O6-alkylguanine-DNA alkyltransferase) is required for reversing specific types of DNA alkylation damage (Singer and Hang, 1997; Pegg, 2000). The primary lesion that Mgmt protects against is O6methylguanine, a lesion that can result in G : C-A : T transitions due to the relatively frequent DNA polymerase misincorporation of thymine opposite O6methylguanine. O4-methylthymine and O6-chloroethylguanine are also substrates for this enzyme, and if unrepaired, can lead to A : T-G : C transitions, and interstrand crosslinks, respectively. The latter base lesion upon resolution through alternate repair pathways may lead to G : C-T : A transversions (Minnick et al., 1993). The Mgmt-mediated repair reaction is stoichiometric and autoinactivating, with each molecule acting only once (reviewed in Pegg, 2000). Consequently, the repair capacity for these types of alkylations is a function of the available Mgmt molecules in the nucleus. The cellular content of Mgmt varies widely from one cell type to another, between tissues, and between individuals (Gerson et al., 1986; Janssen et al., 2001). Furthermore, Mgmt levels have been shown to be either elevated or reduced in specific malignancies, raising the possibility of a phenotype of alkylation resistance, or sensitivity, respectively (reviewed in Gerson, 2002; Mukai and Sekiguchi, 2002). Although considerable attention has been focused on Mgmt owing to the frequent use of alkylating agents in chemotherapy regimens (reviewed in Gerson, 2002), what is the evidence that Mgmt plays a role in the protection of cells under normal circumstances? Hinting at its importance, Mgmt is present in organisms as widely divergent as archaebacteria and mammals. This evolutionary conservation clearly indicates a need to protect against alkylating species such as might be generated as a result of conserved metabolic pathways and/or exposure to some ubiquitous environmental agent (reviewed in Bignami et al., 2000). The loss of Mgmt orthologs in Escherichia coli (ada and ogt) or yeast (MGT1) result in a small increase in the spontaneous mutant frequency (Mf), but specifically,

Mgmt deficiency does not alter spontaneous mutations LE Sandercock et al

5932

an increase in G : C-A : T transitions (Rebeck and Samson, 1991; Xiao and Samson, 1993; Mackay et al., 1994; Vidal et al., 1998; Allay et al., 1999). In contrast, overexpression of Mgmt was shown to protect against the effects of exogenous alkylating agents and reduced the incidence of spontaneous liver tumors as well as the frequency of G : C-A : T transitions (Nakatsuru et al., 1993; Roth and Samson, 2000; Ishikawa et al., 2001; Jansen et al., 2001; Walter et al., 2001; Zhou et al., 2001). Consistent with Mgmt being an essential defense mechanism, compared to controls, Mgmt/ mice exhibited greatly increased rates of mutagenesis and tumorigenesis following parenteral administration of alkylating agents (Tsuzuki et al., 1996; Sakumi et al., 1997; Glassner et al., 1999). Moreover, spontaneous O6-dG alkylation damage, if present, should be a contributing factor in determining spontaneous mutant frequencies and/or mutational spectrum. Through the use of transgenic mice with a passive, retrievable, forward mutation reporter gene such as lacI, spontaneous tissue-specific mutant frequencies and mutation spectra can be determined (Mirsalis et al., 1993). This approach has been used successfully to examine the effects of deficiency or overexpression of a variety of different DNA repair proteins (Andrew et al., 1997, 2000; Narayanan et al., 1997; Allay et al., 1999; BarossFrancis et al., 2001; Zhou et al., 2001; Mark et al., 2002; Wei et al., 2002) of relevance to Mgmt, the lacI transgenic system has been used for the detection of mutations induced by agents known to create O6alkylguanine lesions. Allay et al. (1999) reported a decrease in N-methyl-N-nitrosourea (MNU)-induced lymphomas in mice overexpressing hMGMT in their thymus and spleen, along with a corresponding decrease in both Mf and G : C-A : T transitions in lacI genes rescued from these thymi. Similarly, Zhou et al. (2001) reported a decrease in both the percentage of G : CA : T transitions in lacI genes recovered from liver DNA, and the incidence of tumors in hMGMT-overexpressing transgenic C3HeB mice, a strain that spontaneously develops hepatic neoplasms. Such studies provided evidence that Mgmt can play an active role in mutation suppression within tissues. Hypothesizing that Mgmt is an important factor in determining the number and nature of spontaneous mutations, we characterized lacI reporter gene (Dycaico et al., 1994) mutant frequencies and spectra in the DNA of Mgmt-deficient liver and small intestine. Our results indicate that Mgmt has little, if any, role in determining the spontaneous mutant frequency or mutational spectrum of lacI reporter genes recovered from two different mouse tissues.

Results Mutant frequencies and mutational spectra of Mgmt/ tissues from young mice To establish whether loss of Mgmt was associated with a spontaneous ‘mutator’ phenotype, lacI mutant frequenOncogene

cies (Mf) were determined for small intestinal epithelial and liver DNA samples from 6 to 8 week old mice. Small intestine was selected since it is a continuously dividing tissue that is exposed to ingested materials and bile. Liver was selected because Mgmt levels are highest at this site (Gerson et al., 1986; Major and Collier, 1998), its role in chemical detoxification and activation, and data showing that Mgmt overexpression reduces G : C-A : T mutations in this tissue (Zhou et al., 2001). We obtained lacI Mf for small intestinal and liver DNA samples from five Mgmt/ mice (Table 1). While Mgmt/ small intestinal epithelium revealed an uncorrected Mf that was about 3.7-fold higher (P ¼ 0.05) than that of our control Big Bluet mice, it was slightly lower than small intestinal DNA controls reported by Arrault et al. (2002); and the Mf of Mgmt/ liver cells was 1.5-fold lower than that of the controls (P ¼ 0.018). All the Mf were low, and despite the statistically significant differences observed in our cohorts, these were all within the Mf range reported for Big Bluet tissues, namely: 1.4–6.7 105 (de Boer et al., 1998; Stuart et al., 2000; Arrault et al., 2002; Hill et al., 2004). Thus, Mgmt deficiency was not accompanied by an overt ‘mutator’ phenotype in our mice. Although Mf above the normal range for Big Bluet tissues were not seen, a qualitative change may have been present. To assess the mutation spectra of the Mgmt-deficient tissues, the sequences of between four and 16 lacI mutants per mouse were analysed. Although the low Mf yielded relatively few lacI mutants, 31 and 48 unique mutations were characterized for intestinal, and liver DNA, respectively. As recurrent lacI mutations may have been clonal in origin, changes found more than once within the same animal were eliminated from the analysis. It should be noted that recurrent lacI mutations potentially constitute ‘hotspots’, which we define as a lacI mutation that is observed in more than one animal. To determine the effect of correcting for clonality, spectra were recalculated to include recurrent mutations and compared to the corrected versions via the Monte Carlo estimates of the P-value of the hypergeometric test (abbreviated here as MCA) (Adams and Skopek, 1987; Cariello and Gorelick, 1996; Cariello et al., 1997). No difference was evident (P ¼ 1.0 for liver, and 0.99 for small intestinal epithelium). The corrected mutation spectra of Mgmt-deficient tissues (Table 2) revealed a predominance of transitions (45–60%). G : C-A : T transitions represent 93% of the total transitions in both organs: 74–77% of these occurred at CpG sites (1–3% of total lacI CpG sites) and 8–11% of these were at GpG sites (0.1–0.2% of total lacI GpG sites). The majority of transversions, 84%, were G : CT : A changes. Finally, 13–23% were insertions and deletions, most commonly þ 1/1 frame-shifts. Comparisons of the mutation spectra from the different genotypes by MCA revealed no statistically significant difference (P ¼ 0.4770.02) between the two Mgmt/ spectra, nor between that of Mgmt/ and the Big Bluet database (P ¼ 0.7570.02 for small intestinal epithelial cells; and P ¼ 0.6870.03 for liver). Thus, we found no evidence indicating that Mgmt deficiency altered the


5933 Table 1

Spontaneous lacI mutant frequencies for DNA obtained from small intestine epithelium and liver of 6–8 week-old Mgmt-deficient and wild-type mice Total PFU

Number of mutants

Mf (105)

Clonality %

Mfa (105)

Mgmt/ small intestine epithelium 1 47 F 2 51 F 3 51 F 4 47 M 5 49 F

397 003 213 701 214 824 248 674 263 680

16.00 4.00 6.00 18.00 7.00

4.0 1.9 2.8 7.2 2.7

0.0 33.3 0.0 58.8 0.0

4.0 1.3 2.8 3.0 2.7

Avg s.d.

49 2

267 576 75 513

10.2 6.3

3.7 2.1

18.4 26.8

2.7 1.0

Mgmt/ liver 1 2 3 4 5

51 49 49 49 49

422 782 436 147 593 737 432 970 405 647

13.00 11.00 12.00 9.00 13.00

3.1 2.5 2.0 2.1 3.2

0 0 0 16.7 0

3.1 2.5 2.0 1.7 3.2

Avg s.d.

49 0.9

458 257 76 664

11.6 1.7

2.6 0.6

3.3 7.5

2.5 0.6

Wild-type small intestine epitheliumb 1 41 F 2 41 F 3 41 M 4 41 M 5 41 M

25 4220 220 540 203 160 225 320 209 160

6 2 1 1 2

2.4 0.9 0.5 0.4 1

ND ND ND ND ND

ND ND ND ND ND

Avg s.d.

41 0

222 480 19 811

2.4 2.1

1.0 0.8

Wild-type liver 1 2 3 4

49 49 49 49

ND ND ND ND

ND ND ND ND

Avg s.d.

49 0

Name

Age (days)

Sex

F F M M F

M M M F

35 4920 201 316 227 791 164 519 237 137 82 698

11 9 9 6 8.8 2.1

3.1 4.5 4.0 3.7 3.8 0.6

PFU ¼ plaque forming unit; Mf ¼ mutant frequency; Avg ¼ average; s.d. ¼ standard deviation; ND ¼ not determined. Bold values indicate average and standard deviation. aCorrected for clonality. bFrom Mark et al. (2002)

normal mutational spectrum of mutant lacI transgenes recovered from these two tissues. Mutant frequencies and mutational spectra of Mgmt/ tissues from 12 month-old mice As O6-guanine alkylation-induced mutations would be predicted to accumulate in progenitor cells as a function of time, DNA samples from the livers and small intestines of 12 month-old Mgmt/ mice were analysed (Table 3). While the Mgmt/ small intestinal epithelium lacI Mf was approximately sixfold higher than that of 6–8 week-old mice, it was still within the normal range of most 12 month-old Big Bluet mouse tissues: 4.1970.56 for brain; 8.3971.22 for liver and 16.572.2 for bladder (Stuart et al., 2000). Similarly, the Mf of 12 month-old Mgmt/ liver was fivefold higher than for the 6–8 weekold mice. Although not significantly higher than that of age-matched Big Bluet liver controls from Stuart et al. (2000) (Mf of 8.3971.22), it was significantly higher than the 10 month-old Big Bluet liver controls from Hill et al. (2004) (Mf of 3.171). Another feature was that the

mutation frequencies exhibited a greater variability than those of the other investigators. As mutation spectra can reveal subtle changes that are not reflected by Mf data, we determined the sequences of 10–25 lacI mutants per mouse. In total, 73 and 81 unique mutants were characterized from 12 month-old Mgmt/ small intestinal epithelium and liver, respectively. The spectra, corrected for clonality, are shown in Table 4. Mgmt-deficient tissues revealed a predominance of transitions (45–60%). In all, 90% of these were G : C-A : T transitions, with approximately 75% of these occurring at CpG sites (1–3% of total CpG sites in lacI), and 8–11% at GpG sites (0.1–0.2% of total GpG sites in lacI). The majority of transversions were G : CT : A changes (68–76%). Insertions and deletions (10–16%) were primarily þ 1/1 frame-shifts. When the spectra were recalculated to include recurrent mutations and compared against the corrected spectra using MCA (Adams and Skopek, 1987; Cariello and Gorelick, 1996; Cariello et al., 1997), no difference was evident (P ¼ 1.0 for both tissues). Mutation spectra among the different genotypes were compared by MCA. Oncogene


5934 Table 2

Spontaneous lacI mutation spectra of Mgmt/ small intestine epithelium and liver DNA from 6 to 8 week-old mice compared to spontaneous lacI mutation spectrum from 1.5-month old Big Bluet mice

Classes

Mgmt/

Mgmt/

Wild-type

Small intestinal epithelium

Liver

Livera

Number

% of total

Number

% of total

Number

% of total

14 13 10 (77%) 1 (8%) 1

45 42

60 56 4

36 33 27 (82%) NR 3

54 49 4

3

29 27 20 (74%) 3 (11%) 2

10 7 1 2 0

32 23 3 6 0

12 10 0 1 1

25 21 0 2 2

25 18 1 5 1

37 27 1 7 1

Insertions/deletions Deletion 1 Insertion +1 Deletion 41 Insertion 41

7 3 0 2 2

23 10 0 6 6

6 2 2 2 0

13 4 4 4 0

6 0 3 3 0

9 0 4 4 0

Complex Multi-bp

0 0

0 0

1 0

2 0

0 0

0 0

31

100

48

100

67

100

Transitions G : C-A : T CpG (% of G : C-A : T) GpG (% of G : C-A : T) A : T-G : C Transversions G : C-T : A G : C-C : G A : T-T : A A : T-C : G

Total

NR ¼ not reported. Bold values indicate class totals. aSpectrum obtained from Stuart et al. (2000) Table 3 Spontaneous lacI mutant frequencies for DNA obtained from small intestinal epithelium and liver cells of 12 month-old Mgmt-deficient and wild-type mice Name

Age (months)

Sex

Total PFU

Number of mutants

Mf (105)

Clonality %

Mfa (105)

Mgmt/ small intestinal epithelium 1 12 F 2 12 M 3 12 M 4 12 F Avg 12 s.d. 0

182 580 96 945 106 407 134 892 130 206 38 461

35 14 42 23 28.5 12.5

19.2 14.6 39.5 17.1 22.5 11.5

5.3 23.1 16.7 13.0 14.5 7.4

18.2 11.1 32.9 14.8 19.3 9.5

Mgmt/ liver 1 2 3 4 Avg s.d.

233 307 311 270 274 383 237 559 264 130 36 239

39 29 29 46 35.8 8.3

16.7 9.3 10.6 19.4 14.0 4.8

20.7 4.8 10.5 4.5 10.1 7.6

13.3 8.9 9.5 18.5 12.5 4.4

NR NR

NR NR

8.4 1.2

12 12 12 12 12 0

Wild-type liver from four male miceb Avg 12 s.d. 0

F M M F

1990 000 NR

167a NR

PFU ¼ plaque forming unit; Mf ¼ mutant frequency; Avg ¼ average; s.d. ¼ standard deviation; NR ¼ not reported. Bold values indicate average and standard deviation. aCorrected for clonality. bFrom Stuart et al. (2000)

No statistically significant difference (P ¼ 0.8370.02) between the spectra of the two 12 month-old Mgmt/ tissues was observed, nor between that of the 6–8 week and 12 month-old mice (P ¼ 0.7470.02, and 0.8270.02 for intestinal epithelium, and liver, respectively). There was also no significant difference between old Mgmt/ and Big Bluet mice, although this was the least similar (P ¼ 0.4070.02) for liver. These results suggest that mutations resulting from O6-guanine alkylations do Oncogene

not accumulate in liver or small intestinal cells over time. Analysis of gene mutations in Mgmt/ mice defective in DNA mismatch repair As we observed no significant effect on either Mf or spectra in the absence of Mgmt, we next diminished the capacity for postreplicative repair by ‘removal’ of Msh6,


5935 Table 4 Spontaneous lacI mutation spectra of Mgmt/ small intestine epithelium and liver DNA from 12 month-old mice compared to an agematched spontaneous lacI mutation spectrum from Big Bluet mice

Classes

Mgmt/

Mgmt/

Wild-type

Small intestinal epithelium

Liver

Livera

Number

% of total

Number

% of total

Number

% of total

38 33 25 (75%) 1 (3%) 5

52 45

52 48 4

66 50 31 (62 %) NR 16

44 34

7

42 39 23 (59%) 9 (23%) 3

Transversions G : C-T : A G : C-C : G A : T-T : A A : T-C : G

21 16 2 3 0

29 22 3 4 0

28 19 5 3 1

35 23 6 4 1

55 34 8 7 6

37 23 5 5 4


12 8 0 4 0

16 11 0 5 0

8 3 1 4 0

10 4 1 5 0

22 8 6 5 3

15 5 4 3 2

1 1

1 1

1 2

1 2

3 3

2 2

73

100

81

100

149

100

Transitions G : C-A : T CpG (% of G : C-A : T) GpG (% of G : C-A : T) A : T-G : C

Complex Multi-bp Total

11

NR ¼ not reported. Bold values indicate class totals. aSpectrum obtained from Stuart et al. (2000)

a component of the Msh2 : Msh6 heterodimer, MutSa. This complex recognizes and initiates the repair of base : base mispairs as well as small insertions and deletions (Jiricny and Nystrom-Lahti, 2000). It has a high affinity for G : T mismatches, including O6-methylguanine : T mismatches. MutSa binding to O6-methylguanine : T mismatches can also trigger apoptosis (Hickman and Samson, 1999), primarily under conditions of high mutational load (reviewed in Kaina et al., 2001). In view of the data obtained with Mgmt/ mice we did not expect alkylation loads sufficient to induce apoptosis. In any event, Msh6/ cycling cells exposed to alkylation stress would be predicted to be resistant to apoptosis, and lack of G : T repair would result in increased G : C-A : T mutations in daughter cells. Indeed, using the lacI system, we previously reported that exposure of Msh2-deficient mice to an SN1 alkylating agent caused a dramatic induction of G : C-A : T mutations (Andrew et al., 1998). However, despite a 20-fold increase in lacI Mf over Mgmt/ small intestinal cells, Mgmt/Msh6/ intestinal cells demonstrated a Mf not statistically different from that of Msh6/ mice (P ¼ 0.50). Similarly, while showing a 16fold Mf increase over Mgmt/ liver samples, Mf of Mgmt/Msh6/ mice were not significantly different from those of Msh6/ mice (P ¼ 0.80) (Table 5). The lacI mutational spectrum for both intestinal epithelium and liver was then obtained by sequencing 23–29 and 15–19 mutants per mouse, respectively. In total, 79 mutants were characterized for Mgmt/Msh6/ intestine, and 52 for liver. Correction was made for possible clonality. However, even when the recurrent

mutations were included there was no significant difference from the corrected spectra (P ¼ 0.98 liver; 0.99 intestine). The spectrum for Mgmt/Msh6/ mice (Table 6) was dominated by transitions (71.2–75.9%). G : C-A : T, accounted for 90–97.3% of these, with 55– 66.7% at CpG sites (2.5–6.6% of total lacI CpG sites), and 16.7–30% at GpG sites (0.1–4.8% of total lacI GpG sites). Transversions (13.5–16.5% of the total mutations) were primarily G : C-T : A, and A : T-T : A (representing 57.1–61.5%, and 23.1–42.9%, respectively). Insertions and deletions ranged from 7.6 to 13.5% of the total mutations and were dominated by 1 deletions. Interestingly, Mgmt/Msh6/ mutation spectra from both organs showed roughly a 10% decrease in transitions (compared to the Msh6/ controls), with an increase in total transversions and IDLs. MCA was used to compare the Mgmt/Msh6/ spectra with that of the appropriate control genotypes. Significant differences were seen only between the Mgmt/Msh6/ and the Big Bluet controls (from the database), and between Mgmt/Msh6/ and Mgmt/ intestinal cells (Po0.05). Thus, even in the absence of both Mgmt and Msh6, we found no evidence of changes in either the lacI Mf or spectrum that would be consistent with O6guanine alkylation-induced mutations. Discussion Given the conservation of Mgmt across widely divergent organisms, it could be hypothesized that endogenously generated and/or exogenous alkylating agents continuOncogene


5936 Table 5 Spontaneous mutant frequencies for DNA obtained from small intestinal epithelium and liver of Mgmt/Msh6/ and Msh6/ 6–8 weekold mice Name

Age (days)

Mgmt/Msh6/ 1 2 3 Avg s.d. Mgmt/Msh6/ 1 2 3 Avg s.d.

Sex

small intestine epithelium 56 M 49 F 49 M 51 4 liver 56 49 49

M F M

51 4

Total PFU

Number of mutants

Mf (105)

Clonality %

Mfa (105)

200 172 238 806 233 622

151 145 192

75.4 60.7 82.2

44.4 37.2 11.5

41.9 38.1 72.7

224 200 20 970

162.7 25.6

72.8 11.0

31.1 17.3

50.9 19.0

207 940 217 320 263 797

112 73 91

53.9 33.6 34.5

48.6 25.0 10.0

27.7 25.2 31.0

40.6 11.5

27.9 19.5

28.0 2.9

31.3 43.5 66.1 104.7 56.4 100.7

35.0 48.0 28.0 46.0 19.0 45.0

20.3 22.6 47.6 56.5 45.6 55.4

229 686 29 911

92.0 19.5

Msh6/ small intestine epitheliumb 1 48 F 2 46 F 3 46 M 4 55 F 5 49 F 6 49 M

249 245 200 215 298 126 263 674 218 297 228 514

78 87 197 276 123 230

Avg s.d.

243 012 35 088

165 81.2

67.1 30.0

37.0 12.0

41.3 16.0

172 841 257 220 230 600

52 136 107

30.1 52.9 46.4

30.8 31.3 52.0

20.8 36.4 22.3

43.1 11.7

38.0 12.1

26.5 8.6

49 3

Msh6/ lacI+ liver 1 49 2 55 3 50 Avg s.d.

51 3

F F F

220 220 43 136

98.3 42.7

PFU ¼ plaque forming unit; Mf ¼ mutant frequency; Avg ¼ average; s.d. ¼ standard deviation. Bold values indicate average and standard deviation. aCorrected for clonality. bFrom Mark et al. (2002)

ally challenge the genomes of all species (reviewed in Bignami et al., 2000). In keeping with this, it was reported that Mgmt-deficient E. coli and yeast demonstrated increased spontaneous mutational rates (Rebeck and Samson, 1991; Xiao and Samson, 1993; Mackay et al., 1994; Vidal et al., 1998). In contrast, we found that lacI Mf were not particularly elevated in two Mgmt/ mouse tissues. Although Mgmt-deficient intestinal epithelial DNA from 6 to 8 week-old mice showed a statistically significant increase in Mf over that of control Big Bluet mice, this was still within the range of Big Bluet control tissues, and in fact was lower than that reported for control intestinal DNA by Arrault et al. (2002) (4.871.5 105). Given that we sampled only two tissues, the possibility remains that other Mgmt/ tissues might show significant lacI Mf inductions. Much information can be garnered by analysing mutation spectra, even when Mf are not overtly increased, provided there is a significant increase in DNA lesion- or repair-specific (‘hallmark’) mutations. As mentioned previously, the primary substrates for Mgmt are O6-alkylguanine and O4-alkylthymine (Singer and Hang, 1997), which can lead to G : C-A : T Oncogene

transitions for O6-methylguanine, G : C-T : A transversions for O6-chloroethylguanine (Minnick et al., 1993) and T : A-C : G transitions for O4-methylthymine. Analysis of spontaneous mutations in Mgmt-deficient E. coli, yeast and CHO cells support the importance of O6-alkylguanine in the genesis of elevated levels of G : C-A : T transitions (Rebeck and Samson, 1991; Xiao and Samson, 1993; Mackay et al., 1994; Vidal et al., 1998). Interestingly, while the latter dominated the mutational spectrum, a sensitive reversion assay (Mackay et al., 1994) revealed increases in all classes of base substitutions, except for G : C-T : A. Furthermore, Allay et al. (1999) reported a decrease in mutant frequency, G : C-A : T transitions and lymphomas in thymi of mice overexpressing hMGMT that had been treated with MNU. Zhou et al. (2001) also found overexpression of hMGMT decreased G : C-A : T transitions in male C3HeB mice, a strain susceptible to spontaneous hepatocellular carcinomas hypothesized to arise from chronic alkylation stress. We anticipated that C57BL/6 Mgmt-deficient mice would show an increased proportion of G : C-A : T transitions compared to control mice. This proved not to be the case for either tissue analysed. However, as


5937 Table 6

Spontaneous lacI mutation spectra from Mgmt/Msh6/ and Msh6/ small intestine epithelium and liver DNA of 6–8-week-old mice Mgmt/Msh6/ SI

Classes

Mgmt/Msh6/ liver

Msh6/ SIa

Msh6/ liver

Number

% of total

Number

% of total

Number

% of total

Number

% of total

60 54 30 (56%) 14 (26%) 6

75.9 68.4

71.2 69.2

11

34 29 21 (72%) 4 (14%) 5

87 73

1.9

68 59 34 (58%) 14 (24%) 9

84 73

7.6

37 36 22 (61%) 4 (11%) 1

13 8 1 3 1

16.5 10.1 1.3 3.8 1.3

7 4 0 3 0

13.5 7.7 0.0 5.8 0.0

6 4 0 1 1

7 5 0 1 1

3 2 1 0 0

8 5 3 0 0


6 6 0 0 0

7.6 7.6 0.0 0.0 0.0

7 5 2 0 0

13.5 9.6 3.8 0.0 0.0

7 5 1 0 1

9 6 1 0 1

1 1 0 0 0

3 3 0 0 0

Complex Multi-bp

0 0

0 0

0 1

0.0 1.9

0 0

0 0

1 0

3 0

79

100

52

81

100

39

100

Transitions G : C-A : T CpG (% of G : C-A : T) GpG (% of G : C-A : T) A : T-G : C Transversions G : C-T : A G : C-C : G A : T-T : A A : T-C : G

Total

100

13

SI ¼ small intestine epithelium cells. Bold values indicate class totals. aFrom Mark et al. (2002)

lack of a significant difference does not necessarily imply a significant similarity (Rogozin et al., 2001), the data was inspected for trends. A trend towards an increase in G : C-A : T transitions was observed in the Mgmtdeficient liver compared to intestine (56 vs 42%). As Mgmt levels are highest in liver (Gerson et al., 1986; Major and Collier, 1998), this increase is consistent with the idea that high Mgmt levels could be related to increased alkylation stress in this tissue. Mutations due to alkylating agent exposure would be predicted to accumulate in DNA over time, perhaps to a greater extent in highly self-renewing tissues such as the intestine, where mutations would accumulate within progenitor (stem cell) populations. Consistent with this idea, lacI Mfs increase with age in liver and bladder, but to a lesser degree, in brain (Stuart et al., 2000). It is also possible that alkylation stress increases with age – for example, the level of O6-methylguanine was higher in maternal leukocytes than in cord blood cells (Georgiadis et al., 2000). Thus, as we observed no large differences in either the Mf or spectra of Mgmt deficient vs control mice at 6–8 weeks of age, 12 month-old mice were analysed. Mf increased over younger mice as previously reported for Big Bluet controls (Stuart et al., 2000; Hill et al., 2004). However, in contrast to younger mice, greater variability of mutant frequencies in aged Big Bluet mice have been reported. For example, Stuart et al. (2000) found a Mf of 8.3971.22 for 12 month-old liver DNA, and 14.271.13 for 25 month-old liver DNA, while Hill et al. (2004) reported a Mf of 3.171 for 10 month-old liver DNA, and 7.970.9 for 25 month-old liver DNA. Despite these Mf differences, the mutational spectra were not statistically different in these two reports. Performing MCA (Cariello et al., 1997) on the

12-month-old Stuart et al. (2000) liver spectrum and the composite Hill et al. (2004) liver spectrum, we found no statistically significant difference (P ¼ 0.45), indicating that there was little, if any, interlab variation when it comes to mutational spectrum analysis. Comparisons of the mutation spectra of the 12 month-old Mgmtdeficient mice did not reveal any differences attributable to Mgmt deficiency. If the apparent elevation in mutant frequencies of our 12 month-old Mgmt-deficient mice had been the result of the Mgmt deficiency, then an increase in G : C-A : T transitions should have been apparent. As this was not the case, we were not able to attribute the apparent elevation of Mf to Mgmt deficiency. In summary, employing the lacI mutational reporter to analyse liver and small intestinal DNA of mice, we found no evidence of excess mutations that could be attributed to O6-guanine alkylation events. As the spectra of Mgmt-deficient mice was similar to that of controls, a more sensitive in vivo ‘assay’ was designed by generating mice deficient in both Mgmt and the DNA mismatch repair protein Msh6, a component of MutSa. A similar strategy was employed with Mgmt/ Mlh1/ and Mgmt/Mlh1 þ / mice to demonstrate how the response to an exogenous alkylating agent could be exquisitely regulated (Kawate et al., 1998, 2000). As anticipated from previous results with Msh6deficient mice (Mark et al., 2002), lacI Mf of Mgmt/ Msh6/ mice were considerably higher than those of Mgmt-deficient or control mice, but not Msh6-deficient mice. The Msh6-deficient liver spectrum, like that of the small intestinal epithelial cells, was dominated by G : C-A : T transitions as would be predicted from previous in vitro and in vivo studies (Jiricny and Oncogene


5938

Nystrom-Lahti, 2000; Mark et al., 2002). Given the substrates of Mgmt (Singer and Hang, 1997), we anticipated that the Mgmt/Msh6/ spectra might show a further increase in G : C-A : T transitions, and possibly certain transversions, when compared to the Msh6-deficient spectra. However, there was no significant difference, only a trend towards a greater percentage (97%) of G : C-A : T transitions as a percentage of the total transitions in Mgmt/Msh6/ livers. There was also a trend towards increased total transversions for both tissues in Mgmt/Msh6/ mice which is consistent with the increase in spontaneous transversions of nearly all the categories seen by Mackay et al. (1994) in ogt ada E. coli. Importantly, two studies using DNA mismatch repair-deficient mammalian cell lines reported an increase in A : T-T : A transversions in addition to the A : T-G : C transitions using O4-methylthymine- or O4ethylthymine-containing shuttle vectors (Klein et al., 1990; Pauly and Moschel, 2001). It was primarily the A : T-T : A class that was elevated in the Mgmt/ Msh6/ mice. Vidal et al. (1998) also noted a bias towards G : C-A : T changes at the 50 G of a PuG in ogt ada E. coli; GpG sites being the primary targets of SN1 alkylating agents. No bias towards GpG sites was evident in our data, suggesting that mechanisms other than SN1 alkylation might be at play in determining the lacI spectrum observed. To understand the lack of change in Mf and mutation spectra in Mgmt-deficient animals, it is essential to look at candidates for endogenous alkylating agents. These have included the methyl donor S-adenosylmethionine (SAM), as well as products of lipid peroxidation and amine nitrosation (reviewed in Major and Collier, 1998). SAM, despite being a methyl group donor in the enzymatic methylation of DNA (at CpG sites), and capable of noncatalysed methylation (Lindahl, 1993), is not a likely candidate for O6-dG methylation (Bawa and Xiao, 1999; Posnick and Samson, 1999). Lipid peroxidation products, including compounds capable of DNA oxidation and alkylation, can be generated upon reaction of membrane lipids with free radicals (Vaca et al., 1988; Chung et al., 1996; Nair et al., 1999; Marnett, 2000). Amines such as methylamine and methylurea, amino acids, peptides or polyamines can also be nitrosated to yield moieties that potentially alkylate DNA (Vidal et al., 1998). In studies on the variety of mutations seen to be elevated in alkytransferase-deficient E. coli, Mackay et al. (1994) suggested that in addition to simple methylation reactions, there were agents capable of more complex alkylations. It is possible that nitrosated amines, particularly those generating bulky alkylated DNA adducts (Taverna and Sedgwick, 1996; Sedgwick, 1997; Vidal et al., 1998), are involved in bacteria. Nitrosated amines may also be produced within mammalian systems. For example, the direct chemical nitrosation of secondary amines by nitrite could be caused either by enzymes originating from intestinal flora, or spontaneously, in the low pH environment of the stomach, from dietary nitrates and nitrites (reviewed in Benjamin, 2000). Furthermore, high concentrations Oncogene

of nitric oxide released by macrophages during inflammatory responses, could also lead to nitrosation (reviewed in Espey et al., 2002). For example, O6methylguanine and urinary N-nitroso compound levels are both elevated in the bladders of patients with schistosomiasis, an infection associated with inflammation and cancer predispoition (reviewed in Mostafa et al., 1999). In keeping with a role for Mgmt in this context, a number of reports have provided in vivo evidence for Mgmt having a role in the prevention of MNU and dimethylnitrosamine-induced tumors in mice (Allay et al., 1997, 1999; Iwakuma et al., 1997; Glassner et al., 1999; Reese et al., 2001). Another by-product of high NO concentrations is lipid peroxidation (Radi et al., 1991; Hogg et al., 1993; Virag et al., 2003), which could yield alkylating species, analogous to the lipid peroxidation-derived etheno-DNA adducts, that may play a role in cancer initiation within foci of chronic inflammation (Nair et al., 1998a, b, 1999). Why was Mgmt deficiency not associated with a mutator phenotype? Perhaps another system, such as nucleotide excision repair or the presence of a second methyltransferase, is involved in repairing the low levels of spontaneous alkylation damage that may occur in vivo. The latter is a possibility given the very low levels of residual O6-methylguanine repair activity detected in the livers of Mgmt deficient mice (Glassner et al., 1999). It is also possible that despite Mgmt being highly conserved in higher organisms that this molecule is not required to protect cells against low levels of guanine alkylation. Since inflammation represents a potential source of alkylating species, it would be of considerable interest to subject Mgmt-deficient mice to a chronic inflammatory challenge to determine whether this stimulus would produce a Mf increase and/or a shift in the mutational spectrum. Lastly, it is conceivable that the lacI reporter transgene array we used to assay mutations is located within a region of chromatin that is not readily accessible to low concentrations of putative endogenous alkylating agents. Further investigation is clearly warranted to establish why Mgmt deficiency does not significantly alter spontaneous mutagenesis in mice. Materials and methods Transgenic mouse lines Msh6 (Edelmann et al., 1997) and Mgmt deficient (Glassner et al., 1999) mice were crossed with Big Bluet mice (Stratagene, La Jolla, CA, USA) and then with each other in order to generate Mgmt/Msh6/lacI þ mice and all other possible genotypes. Mice were on a C57BL/6 background. They were housed in microisolator-type cages at 251C with a 12-h light/dark cycle in a (viral antibody-free) barrier facility in accordance with University of Calgary Animal Care Committee guidelines. Irradiated, pelleted Mouse Diet (Purina Picolab #5058) and water were given ad libitum. Genotype testing Msh6, Mgmt and lacI genotyping was carried out by PCR using DNA extracted from mouse tail tips as previously described (Glassner et al., 1999; Mark et al., 2002).


5939 Tissue extraction and genomic DNA isolation

Data analysis and databases

Mice were euthanized by carbon dioxide inhalation. Tissues, with the exception of small intestine, were immediately extracted and flash-frozen in liquid nitrogen and then stored at 801C. Small intestinal epithelial cells and DNA were isolated as previously described (Baross-Francis et al., 2001). Liver DNA isolation was carried out using RecoverEaset (Stratagene, La Jolla, CA, USA) according to the manufacturer’s instructions.

The Monte Carlo estimation of the P-value of the hypergeometric test was used to make pair-wise statistical comparisons of 12 mutant categories from the various genotypes (Adams and Skopek, 1987; Cariello and Gorelick, 1996; Cariello et al., 1997). The program was run with 1700 iterations. Significance was set at a ¼ 0.05. Student’s t-test and other statistics were performed using Microsoft Excel. lacI mutation databases were obtained from Mutations and Mutant Frequencies in Big Bluet (http://eden.ceh.uvic.ca/ results.htm) and Transgenic-Bacterial lacI database and MutaBase Software (http://www.ibiblio.org/dnam/des_laci.htm) (Cariello and Gorelick, 1996; Cariello et al., 1997). The small intestinal epithelial cell mutation database was based on data from Quillardet et al. (2000), and spontaneous mutations in age-matched liver were from Stuart et al. (2000).

Determination of lacI gene mutant frequency and mutation spectrum Transgenic l-phage rescue procedures were as described in the standardized Big Bluet protocol (Stratagene). Mutant frequencies were calculated by determining the ratio of mutant (blue) to wild-type (clear) plaques. For each mouse tissue DNA sample, a minimum of 100 000 plaques were enumerated, except for Mgmt/ lacI þ liver cells, where at least 400 000 plaques were counted. Blue plaques were verified by replating as described (Andrew et al., 1996), and randomly selected mutants were then isolated for lacI gene sequence analysis. Mutants were sequenced at two different facilities. At the Center for Molecular Medicine and Therapeutics (University of British Columbia) DNA Sequencing Core Facility, an ABI 377 Perkin–Elmer instrument was employed. Plaque DNA samples were first amplified using primers 11 (50 -GACACCATCGAATGGTGC-30 ) and 1201 (50 -ACAATTCCACACAACAT-30 ) and then purified using QIAquicks PCR DNA Purification Kit (QIAGEN, Valencia, CA, USA). Sequencing was carried out using primers F1 (50 GACACCATCGAATGGTGC-30 ), F2 (50 -GCTGCCTGCACTAATGTTCCG-30 ), R1 (50 -CTGGTCAGAGACATCAAG-30 ), R2 (50 -ATCGTCGTATCCCACTACCG-30 ), R3 (50 -ACAATT CCACACAACAT-30 ). These sequences were analysed with MultAlin software (Corpet, 1988). At the DNA Sequencing Core Facility (University of Victoria), DNA was isolated from plaques and then sequenced using a LI-COR 4200 automated sequencer with primers 234 (50 -GCG TCG ATT TTT GTG ATG CT-30 ) and 1337 (50 -CGC TAT TAC GCC AGC TGG-30 ). Alignment was carried out using SeqMan II, version 5.03 from DNASTAR Inc.

Abbreviations IDLs, insertion/deletion loops; MCA, Monte Carlo analysis; Mf, mutant frequency; Mgmt, O6-methylguanine-DNA methyltransferase; MMR, mismatch repair; MSH, MutS homolog. Acknowledgements We are grateful to W Edelmann for the Msh6-deficient mice, and to S Lines and S Chan for generating the various genetic crosses and caring for the animal colonies. This work was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society, and by an Alberta Heritage Foundation for Medical Research (AHFMR) establishment grant (both to FRJ). LES and HAL were supported by AHFMR post-doctoral training awards, and FRJ was the recipient of AHFMR Medical Scientist and Canada Research Chair awards. LDS is the Ellison American Cancer Society Research Professor. Generation of the Mgmt-null mouse was supported by National Institutes of Health Grants CA75576 and CA55042 (to LDS).

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