Introduction

The mammalian protein MBD4 contains a methyl-CpG binding domain (Hendrich and Bird, 1998) and can also enzymatically remove T or U from a mismatched CpG site in vitro (Hendrich et al., 1999; Petronzelli et al., 2000). Recently, we confirmed that MBD4 functions in vivo to minimize the mutability of 5-methycytosine (m⁵C) by showing that the frequency of mutation at m⁵CpG dinucleotides in a murine transgene is significantly increased in Mbd4^-/- mice (Millar et al., 2002). The MBD4 gene has also been found to be mutated in a high frequency of human mismatch repair-deficient colorectal cancers, although this is rarely a biallelic event (Riccio et al., 1999; Bader et al., 1999, 2000). Furthermore, Millar et al. (2002) have shown that deficiency of MBD4 accelerates tumorigenesis on an Apc^Min background. Analysis of these tumours showed an increased frequency of CpG mutation at the Apc allele, but that this was not fully penetrant. This raised the possibility that MBD4 may do more than just initiate repair of TG mismatches.

In addition to its role as a thymine glycosylase, MBD4 has been shown to interact with the MMR protein MLH1 (Bellacosa et al., 1999). To date, the functional basis of this interaction is poorly understood. Since canonical mammalian mismatch repair (MMR) is independent of methylation status and the mutation rate and spectra are very different between Mbd4^-/- mice and MMR-deficient mice (Millar et al., 2002), it appears that the interaction between MMR and MBD4 cannot be simply explained by a role for MBD4 in MMR (Bellacosa, 2001; Drummond and Bellacosa, 2001).

A second possibility is that the MBD4–MMR interaction is associated with the normal damage response. The MMR proteins have been shown to be essential for the normal response to a wide spectrum of agents. These include oxidative damage, ionizing radiation (at least in primary fibroblasts), cisplatin, 6-TG, UV and 5-FU damage (Buermeyer et al., 1999). Indeed the MMR proteins have been shown to bind directly to O6 methylguanine (O6meG) lesions (which are thought to mimic a mismatch as the O6meG pairs with a T) and signal apoptosis either directly or through cycles of futile repair (Karran and Bignami, 1992; Fishel, 1999).

We have previously shown that MMR-deficient mice have a compromised apoptotic response to a range of DNA-damaging agents in vivo including the alkylating agents temozolomide, NMNU and MNNG, cisplatin and nitrogen mustard (Toft et al., 1999; Sansom et al., 2001; Sansom and Clarke, 2002). Here we ask whether MBD4 mediates MMR-dependent apoptosis in the murine small intestine, and furthermore whether MBD4 is a general mediator of the apoptotic response.

Materials and methods

Mice

Mbd4 and Mlh1 mutant animals were derived from a colony segregating for Ola/129 and C57BL6J genomes, but which was had been backcrossed four generations onto the C57BL6J background and so were predominantly (93.75%) C57/BL6J. Mice were genotyped by PCR as previously described (Prolla et al., 1998; Millar et al., 2002), and in all experiments littermate controls were used. To rule out the possibility that differences in the apoptotic response were due to SV129-derived genes linked to the Mbd4 locus, we determined if there was any difference in apoptotic response of purebred 129SV and C57B1/6 mice 6 h following exposure to either cisplatin (mean apoptotic bodies per 50 half cryptss.d. values of 17928.5 and 13134.4, respectively) or temozolomide (1128.5 and 13932.4, respectively). For both agents, genetic background was not found to influence the apoptotic response compared to wild-type out-bred values (cisplatin: Mann–Whitney U-test, P=0.66 for SV129 and P=0.19 for C57B16; temozolomide: Mann–Whitney U-test, P=0.39 for SV129 and P=1.0 for C57B16). Furthermore, the levels of apoptosis seen for either cisplatin- or temozolomide-treated SV129 and C57B16 mice remained significantly higher than those for the out-bred Mbd4 null mice (P<0.04 for all combinations).

Reagents and administration

Mice, 8 to 12 weeks old, were given intraperitoneal (i.p.) cisplatin (10–20 mg/kg), temozolomide (100 mg/kg) and 5-FU (400 mg/kg 2). The two injections of 5-FU were administered 6 h apart according to Pritchard et al. (1998). Cisplatin and 5-FU were obtained from David Bull Laboratories/Faulding Pharmaceuticals, while temozolomode was a gift from Malcolm Stevens. Mice were exposed to -irradiation using two different ¹³⁷Cs sources. These delivered -irradiation at 0.27 Gy/min or at 0. 423 Gy/min. Irrespective of the source used, animals were exposed such that they received a dose of either 5, 10 or 15 Gy.

Quantitation of apoptosis

At each indicated time point following injection, a minimum of three animals were killed and the small intestine removed, flushed with water and fixed overnight in methacam (four parts methanol, two parts chloroform, one part acetic acid). Histological sections were made and apoptosis scored as previously described (Toft et al., 1999). A minimum of 50 half crypts were scored per animal. This method was used in preference to indirect assessments of apoptosis because the apoptotic response within the intestine has previously been well defined using this approach (Potten, 1990; Hendry et al., 1997; Toft et al., 1999). All data were counted in a double-blinded manner.

Microcolony assay of clonogenic survival

The microcolony assay was performed as previously described (Potten, 1990; Hendry et al., 1997). Briefly, 72 h after injection with cytotoxic agents the murine small intestines were removed. The top third was then cut into small pieces and bound into a bundle with 3M surgical tape. These were fixed in 10% formalin and embedded. Histological cross-sections were made and the numbers of surviving crypts were then counted around the circumference of the intestines. Crypts were scored as viable if they contained more than five consecutive live cells. As all mice were harvested at the same time and comparison between genotypes are being made, there was no rationale for applying any correction factor (Ijiri and Potten, 1983). Doses of 15 and 20 mg/kg were used for cisplatin and 400 mg/kg 2 5-FU according to Ijiri and Potten (1983) and Pritchard et al. (1998). All data were counted in a double-blinded manner.

BrdU immunohistochemistry

Mice were injected with 0.25 ml of bromodeoxyuridine (BrdU) (Amersham) 2 h prior to harvesting. The staining was done on paraffin-embedded, methacarn-fixed intestines. Briefly, after a quick wash in water, slides were shaken at 60°C for 10 min in 1 M HCl for antigen retrieval. They were washed in PBS and then blocked for 20 min in 1.5% H₂O₂. Slides were incubated in 1% BSA/PBS for 20 min and then incubated in BrdU conjugate (Roche) diluted to one part in 50. Slides were washed in PBS and then developed in DAB. All data were counted in a double-blinded manner.

Results and discussion

Reduced apoptosis in Mbd4-deficient mice

We analysed the influence of Mbd4 status upon the apoptotic response of intestinal enterocytes following exposure to a range of different cytotoxic agents to fully characterize any reliance upon MBD4 function. For all these agents, we investigated the response 6 h following exposure, as this has previously been shown to report the maximal or near-maximal apoptotic response (e.g. Toft et al., 1999; Sansom and Clarke, 2002). In the case of ionizing radiation, gene deficiency has been linked with a delayed apoptotic response, for example, in the absence of p53 (Toft et al., 1999). Therefore, for this agent we also investigated the levels of apoptosis at 72 h. Mice null for Mbd4 showed a markedly reduced apoptotic response following exposure to ionizing radiation at 6 h (P=0.001), cisplatin (P=0.01) and the alkylating agent temozolomide (P=0.01) (Figure 1). With respect to the 72 h time point, we found no evidence for a delayed wave of apoptosis following ionizing radiation in the absence of MBD4.

Figure 1.

(a) Apoptosis scored per 50 half crypts following 5 Gy -irradiation, 10 mg/kg cisplatin and 100 mg/kg temozolomide treatment. Black bars, wild-type mice; open bars, Mbd4^-/- mice. At least three mice were used for every time point and error bars represent s.d. Mbd4^-/- mice had a significantly reduced apoptotic response at 6 h following all drugs used (-irradiation, n=8, P=0.001, temozolomide and cisplatin n=5, P=0.001). There was no gene dependency at 72 h (P=0.68, n=3). All statistical analyses were performed using the Mann–Whitney U-test. (b) Apoptosis scored per 50 half crypts over a 48 h period following 10 mg/kg temozolomide. Black bars, wild-type mice; open bars, Mbd4^-/- mice. At least three mice were used for every time point and error bars represent s.e.m. (c) Apoptosis scored per 50 half crypts over a 48 h period following 100 mg/kg cisplatin treatment. Black bars, wild-type mice; open bars, Mbd4^-/- mice. At least three mice were used for every time point and error bars represent s.e.m. (d) Representative photograph of apoptosis induced within wild-type intestinal crypts 6 h following exposure to cisplatin. Haematoxylin and eosin stained section, arrows indicate apoptotic bodies. (e) Representative photograph of apoptosis induced within MBD4 deficient intestinal crypts 6 h following exposure to cisplatin. Haematoxylin and eosin stained section, arrows indicate apoptotic bodies

Full figure and legend (233K)

These data therefore establish a role for Mbd4 in mediating the maximal or near-maximal apoptotic response to a series of different cytotoxic agents. To further investigate the kinetics of these responses, we analysed an extended time course for two agents, temozolomide and cisplatin. For temozolomide, a reduction in the apoptotic response was observed at both 6 and 11 h following treatment in the absence of MBD4 (P<0.01; Mann–Whitney U-test). For cisplatin, similar reductions were observed at 6 and 10 h (P<0.04; Mann–Whitney U-test). This extended analysis therefore confirms not only a role for MBD4 in mediating the normal programme of cell death following exposure to a range of DNA-damaging agents, but also shows that significant MBD4-independent apoptosis does occur following exposure to these agents.

One possible interpretation of these data is that MBD4 deficiency leads directly to mutations in other proapoptotic genes, such that frequent somatic mutation impairs the ability to engage apoptosis. Although we cannot formally rule out this possibility, this hypothesis seems unlikely, as the reported elevation in mutation rate in the absence of MBD4 is almost certainly too low to generate sufficient numbers of mutant clones to modulate the apoptotic response (Millar et al., 2002). It seems more likely that MBD4 plays a direct role in mediating apoptosis, a hypothesis supported by the reported interaction between MBD4 and FADD (Screaton et al., 2003).

The observed reduction in the apoptotic response of Mbd4 mutant mice implies that a proportion of cells fail to be appropriately deleted in the absence of MBD4. This in turn implies that clonogenic survival would be increased in Mbd4 null mice. Figure 2 details the results of intestinal microcolony assays following exposure to DNA damage. MBD4 deficiency leads to no observable difference following exposure to 10 and 15 Gy -radiation (Figure 2a). However, increased clonogenic survival was observed following exposure to 15 mg/kg cisplatin (Figure 2b), indicating that Mbd4 status can influence long-term in vivo survival, albeit in a damage-type-dependent manner.

Figure 2.

(a) Clonogenic survival scored following 10 and 15 Gy -irradiation. Black bars, wild-type mice; open bars, Mbd4^-/- mice. At least three mice were used for every time point and error bars represent s.d. MBD4 deficiency did not affect crypt survival at either dose (10 Gy, P=0.66, n=4, 15 Gy, P=0.38). (b) Clonogenic survival following 15 and 20 mg/kg cisplatin treatment. Black bars, wild-type mice; open bars, Mbd4^-/- mice. At least three mice were used for every time point and error bars represent s.d. MBD4 deficiency caused a significant increase in crypt survival following exposure to 15 mg/kg cisplatin (P=0.03, n=6). All statistical analyses were performed using the Mann–Whitney U-test

Full figure and legend (27K)

MBD4 and 5-FU damage

Recently, deficiency of MLH1 has been implicated in resistance to damage by 5-FU, a cytotoxic ribosomal poison (Meyers et al., 2001). In addition, MBD4 has previously been shown to bind to 5-FU damage (Petronzelli et al., 2000). We therefore wished to test whether Mbd4 status was important for the apoptotic response and long-term survival in vivo following exposure to this agent. It should be noted that the normal kinetics of apoptosis following 5-FU differ somewhat from the other agents used here, with the apoptotic response potentially mediated by multiple mechanisms of action, including inhibition of thymidylate synthase, which gives rise to DNA damage, and by incorporation into RNA (e.g. Pritchard et al., 1998). For these reasons the apoptotic response is potentially more complex than following other cytotoxic drugs and we therefore analysed the requirement for MBD4 over an extended time course, and also assessed changes in crypt cellularity and S-phase labelling.

Figure 3a displays the apoptotic response scored over a 48 h timecourse following 2 400 mg/kg 5-FU treatment. MBD4 deficiency causes a markedly reduced apoptotic response at 10 h following drug treatment (P=0.01; Mann–Whitney U-test). We next investigated cell cycle kinetics via incorporation of BrdU at S phase as Pritchard et al. (1998) argued that increased enterocyte survival (in p53-deficient animals following 400 mg/kg 5-FU treatment) is determined by changes in both apoptosis and proliferation. We found significantly increased proliferation at 16, 18 and 20 h as measured by BrdU incorporation in the MBD4-deficient mice (Figure 3b). The changes in apoptosis and proliferation resulted in an increased epithelial cell number in the Mbd4^-/- mice at 48 h (Figure 3c) and a significant increase in clonogenic survival (as measured by the microcolony assay) at 72 h (Figure 3d). These results clearly establish a role for MBD4 in the recognition of 5-FU damage.

Figure 3.

(a) Apoptosis per 50 half crypts following 2 400 mg/kg 5-FU treatment. Black bars, wild-type mice; open bars, Mbd4^-/- mice. At least three mice were used for every time point and error bars represent s.d. MBD4 deficiency caused a significant reduction in apoptosis at 10 h following 5-FU treatment (P=0.001, n=8). (b) S phase incorporation per 50 half crypts following 2 400 mg/kg 5-FU treatment. Black bars, wild-type mice; open bars, Mbd4^-/- mice. At least three mice were used for every time point and error bars represent s.d. MBD4 deficiency caused a significant increase in BrdU labelling at 16, 18 and 20 h following 5-FU treatment (P<0.05, n=3). (c) Average epithelial cell number per half crypt. Black bars, wild-type mice; open bars, Mbd4^-/- mice. At least 3 mice were used for every time point and error bars represent s.d. MBD4 deficiency caused a significant increase in epithelial cell number 48 h following 5-FU treatment (P<0.05, n=3). (d) Clonogenic survival of intestinal crypts 72 h following 2 400 mg/kg 5-FU treatment. Black bars, wild-type mice; open bars, Mbd4^-/- mice. At least three mice were used for every time point and error bars represent s.d. MBD4 deficiency caused a significant increase crypt survival following 5-FU treatment (P=0.02, n=6). All statistical analyses were performed using the Mann–Whitney U-test

Full figure and legend (58K)

Apoptosis in mice doubly mutant for Mbd4 and Mlh1

The finding that MBD4 is mutated in a high proportion of RER+ intestinal tumours (Bader et al., 1999, 2000; Riccio et al., 1999), together with the reported physical interaction between MLHl and MBD4 (Bellacosa et al., 1999), prompted us to address the interdependence of MBD4 and MLH1 in signalling apoptosis. We therefore generated mice doubly mutant for Mbd4 and Mlh1. If MBD4 and MLH1 operate in separate pathways, one would predict an additive decrease in the levels of apoptosis. Levels of apoptosis were scored at time points selected to reflect maximal induction of the apoptotic response in singly mutated Mbd4^-/- and Mlh1^-/- mice and also in the double null (Mbd4^-/-, Mlh1^-/-) mice following exposure to 5-FU (Figure 4a), temozolomide (4B), cisplatin (4C) and -irradiation (4D). Following exposure to 5-FU and temozolomide both single mutants showed significantly reduced apoptosis, but this effect was not enhanced by simultaneous mutation of both genes (Figure 4a,b). The failure to see an additive effect argues that, for these agents, MBD4 operates within the same pathway as MLHl-dependent apoptosis.

Figure 4.

(a) Apoptosis per 50 half crypts 10 h following 2 400 mg/kg 5-FU treatment. Black bars, wild-type mice; open bars, Mbd4^-/- mice; grey bars, Mlh1^-/- mice and open bars with diagonal stripes, Mbd4^-/- Mlh1^-/- mice. At least three mice were used for every time point and error bars represent s.d. Both Mbd4 and Mlh1 deficiency caused a significant reduction in apoptosis at 10 h following 5-FU treatment (P<0.01, n=6). The double mutants showed significantly reduced apoptosis compared to wild-type mice (P=0.04, n=3), but no significant reduction compared to either Mlh1^-/- (P=0.38, n=3) or Mbd4^-/- mice (P=0.65, n=3). (b) Apoptosis per 50 half crypts 6 h following 100 mg/kg temozolomide treatment. At least three mice were used for every time point and error bars represent s.d. Black bars, wild-type mice; open bars, Mbd4^-/- mice; grey bars, Mlh1^-/- mice and open bars with diagonal stripes, Mbd4^-/- Mlh1^-/- mice, Mbd4 and Mlh1 deficiency caused a significant reduction in apoptosis at 6 h following cisplatin treatment (P=0.01, n=5). Double mutants showed reduced apoptosis compared to wild-type mice (P=0.04; Mann–Whitney U-test, n=3), but there was no significant reduction compared to either Mlh1^-/- (P=0.765, n=3) or Mbd4^-/- mice (P=0.365, n=3). All statistical analyses were performed using the Mann–Whitney U-test. (c) Apoptosis per 50 half crypts 6 h following 10 mg/kg cisplatin treatment. At least three mice were used for every time point and error bars represent s.d. Black bars, wild-type mice; open bars, Mbd4^-/- mice; grey bars, Mlh1^-/- mice and open bars with diagonal stripes, Mbd4^-/- Mhl1^-/- mice. Mbd4 and Mlh1 deficiency caused a significant reduction in apoptosis at 6 h following cisplatin treatment (P=0.04, n=3). Mbd4^-/- mice showed significantly lower levels of apoptosis compared to Mlh1^-/- mice (P=0.04, n=3). Double mutants showed significantly reduced apoptosis compared to wild-type and Mlh1^-/- mice (P=0.04, n=3), but there was no significant reduction compared to Mbd4^-/- mice (P=0.45, n=3). (d) Apoptosis per 50 half crypts 6 h following 5 Gy -irradiation. These experiments yielded higher overall levels of apoptosis as compared to Figure 1, as a different Cs¹³⁷ source was used with a higher dose rate (see Materials and methods). At least three mice were used for every time point and error bars represent s.d. Black bars, wild-type mice; open bars, Mbd4^-/- mice; grey bars, Mlh1^-/- mice and open bars with diagonal stripes, Mbd4^-/- Mlh1^-/- mice, Mbd4 deficiency caused a significant reduction in apoptosis at 6 h following -irradiation (P=0.00 l, n=4). Mlh1^-/- mice showed no significant reduction compared to wild-types (P=0.45, n=4). Double mutants showed significantly reduced apoptosis compared to wild-type and Mlh1^-/- mice (P=0.01, n=4), but there was no significant reduction compared to Mbd4^-/- mice (P=0.55, n=4)

Full figure and legend (147K)

Following cisplatin the reduction in apoptosis observed in the Mlh1^-/- mice was very small (Figure 4c), similar to that previously observed in the Msh2^-/- mice (Toft et al., 1999). MBD4 deficiency results in a significantly greater impairment of the apoptotic response than MLHl deficiency (P=0.04; Mann–Whitney U-test), suggesting that MBD4 can also mediate MMR-independent apoptosis. This notion is further supported by analysis of the response to ionizing radiation that was shown to be dependent on MBD4 but not MLHl (Figure 4d), although we have not formally ruled out the possibility that other molecules in the MMR system may be involved in this response.

Significance of MBD4-mediated apoptosis

We and others have shown that the failure to engage apoptosis does not necessarily predict long-term intestinal enterocyte survival in vivo, so raising concerns about the precise relevance of failed apoptosis to carcinogenesis (Hendry et al., 1997; Sansom and Clarke, 2000, 2002). However, the finding that MBD4 deficiency leads to both diminished apoptosis and increased clonogenic survival after both cisplatin and 5-FU exposure clearly demonstrates an important role in deleting (presumably damaged) cells following DNA damage. We have previously shown that deficiency of MSH2 does not increase long-term survival following cisplatin damage (as assessed by the microcolony assay, Sansom et al., 2001), again indicating that loss of MBD4 can lead to a more substantial phenotype than MMR deficiency. The failure to see an MBD4-dependent increase in survival following -irradiation is not surprising given the recent findings of Paris et al. (2001), who showed endothelial cell survival is the prime determinant of long-term clonogenic survival in the intestine following -irradiation. Indeed, complete loss of the immediate apoptotic response in the epithelium of p53-deficient mice only weakly influences clonogenic survival following -irradiation (Hendry et al., 1997).

With respect to neoplasia, we recently demonstrated that MBD4 suppresses intestinal tumorigenesis in the Apc^Min/+ mouse (Millar et al., 2002). This suppression may be mediated by its role as a thymine glycosylase or through the novel role we demonstrate here in mediating apoptosis. In support of a mechanistic role for MBD4-dependent apoptosis in tumour suppression, analysis of the tumours arising in the Apc^Min/+, Mbd4^-/- mouse showed enhanced CpG mutability in only a third of the tumours analysed, with in most cases the initially wild-type Apc allele still being lost through loss-of-heterozygosity (LOH). It is therefore possible that in a proportion of tumours MBD4 mediates tumour suppression through mechanisms other than the repair of spontaneous deamination events, such as apoptotic signalling.

In conclusion, we have shown that a significant proportion of the in vivo apoptotic response to a range of cytotoxic agents is reliant upon functional MBD4. The precise mechanism of this reliance is as yet unclear, although it seems likely that it is at least in part mediated through MBD4s interaction with FADD; especially given the finding that cellular stress-induced and DNA damage-induced apoptosis is in part dependent upon functional FADD (Herr et al., 1997; Micheau et al., 1999). This potential mechanism is described and discussed further in the accompanying paper (Screaton et al., 2003). Taken together, our results argue that MBD4 may suppress tumorigenesis not only by suppressing 5 methyl CpG deamination but also by mediating apoptosis in cells characterised by DNA damage.

References

1. Bader S, Walker M and Harrison D. (2000). Br. J. Cancer, 83, 1646–1649. | Article | PubMed | ChemPort |
2. Bader S, Walker M, Heindrich B, Bird A, Bird C, Hooper M and Wyllie A. (1999). Oncogene, 18, 8044–8047. | Article | PubMed | ISI | ChemPort |
3. Bellacosa A. (2001). J. Cell. Physiol., 187, 137–144. | Article | PubMed | ChemPort |
4. Bellacosa A, Cicchillitti L, Schepis F, Riccio A, Yeung AT, Matsumoto Y, Golemis EA, Genuardi M and Neri G. (1999). Proc. Natl. Acad. Sci. USA, 96, 3969–3975. | Article | PubMed | ChemPort |
5. Buermeyer AB, Deschennes SM, Baker SM and Liskay RM. (1999). Annu. Rev. Genet., 33, 533–564. | Article | PubMed | ISI | ChemPort |
6. Drummond JT and Bellacosa A. (2001). Nucleic Acid Res., 29, 2234–2243. | Article | PubMed |
7. Fishel R. (1999). Nat. Med., 5, 1239–1241. | Article | PubMed | ISI | ChemPort |
8. Hendrich B and Bird A. (1998). Mol. Cell. Biol., 18, 6538–6547. | PubMed | ISI | ChemPort |
9. Hendrich B, Hardeland U, Ng HH, Jiricny J and Bird A. (1999). Nature, 401, 301–304. | Article | PubMed | ISI | ChemPort |
10. Hendry JH, Cai WB, Roberts SA and Potten CS. (1997). Radiat. Res., 148, 254–259. | Article | PubMed | ISI | ChemPort |
11. Herr I, Wilhelm D, Bohler T, Angel P and Debatin K-M. (1997). EMBO J., 16, 6200–6208. | Article | PubMed | ChemPort |
12. Ijiri K and Potten CS. (1983). Br. J. Cancer, 47, 175–185. | PubMed | ISI | ChemPort |
13. Karran P and Bignami M. (1992). Nucleic Acid Res., 20, 2933–2940. | PubMed |
14. Meyers M, Wagner MW, Hwang HS, Kinsella TJ and Boothman DA. (2001). Cancer Res., 61, 5193–5201. | PubMed | ISI | ChemPort |
15. Micheau O, Solary E, Hammann A and Dimanche-Boitrel MT. (1999). J. Biol. Chem., 274, 7987–7992. | Article | PubMed | ISI | ChemPort |
16. Millar CB, Guy J, Sansom OJ, Selfridge J, MacDougall E, Hendrich B, Keightley PD, Bishop SM, Clarke AR and Bird A. (2002). Science, 297, 403–405. | Article | PubMed | ISI | ChemPort |
17. Paris F, Fuks Z, Kang A, Capodieci P, Juan G, Ehleiter D, Haimovitz-Friedman A, Cordon-Cardo C and Kolesnick R. (2001). Science, 293, 293–297. | Article | PubMed | ISI | ChemPort |
18. Petronzelli F, Riccio A, Markham GD, Seeholzer SH, Genuardi M, Karbowski M, Yeung AT, Matsumoto Y and Bellacosa A. (2000). J. Biol. Chem., 185, 473–480.
19. Potten CS. (1990). Int. J. Radiat. Biol., 58, 925–973. | PubMed | ChemPort |
20. Pritchard DM, Potten CS and Hickman JA. (1998). Cancer Res., 58, 5453–5465. | PubMed | ChemPort |
21. Prolla TA, Baker SM, Harris AC, Tsao EL, Yao X, Bronner CE, Zheng BH, Gordon M, Reneker J, Amheim N, Shibata D, Bradley A and Liskay RM. (1998). Nat. Genet., 18, 276–279. | Article | PubMed | ISI | ChemPort |
22. Riccio A, Aaltonen LA, Godwin AK, Loukola A, Percesepe A, Salovaara R, Masciullo V, Genuardi M, Paravatou-Petsotas M, Bassi DE, Ruggeri BA, Klein-Szanto AJP, Testa JR, Neri G and Bellacosa A. (1999). Nat. Genet., 23, 266–268. | Article | PubMed | ISI | ChemPort |
23. Sansom OJ and Clarke AR. (2000). Mutation Res.-Fundam. Mol. Mech. Mutagen., 452, 149–162.
24. Sansom OJ and Clarke AR. (2002). Oncogene, 21, 5934–5399.
25. Sansom OJ, Toft NJ, Winton DJ and Clarke AR. (2001). Oncogene, 20, 3580–3584. | Article | PubMed | ISI | ChemPort |
26. Screaton RA, Kiessling S, Sansom OJ, Millar CB, Maddison K, Bird A, Clarke AR, Frisch SM. (2003). Proc. Natl. Acad. USA, 100, 5211–5216.
27. Toft NJ, Winton DJ, Kelly J, Howard LA, Dekker M, Riele HT, Arends MJ, Wyllie AH, Margison GP and Clarke AR. (1999). Proc. Natl. Acad. USA, 96, 3911–3915.

Acknowledgements

We thank Nathan Hill for maintenance of animal stocks and Steven Frisch for helpful discussions. This work was supported by Grants from the Cancer Research UK and the Wellcome trust.