British Medical Bulletin Advance Access originally published online on February 1, 2008
British Medical Bulletin 2008 85(1):17-33; doi:10.1093/bmb/ldm036
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O6-Methylguanine-DNA methyltransferase inactivation and chemotherapy
Paterson Institute for Cancer Research, University of Manchester, Manchester, UK
* Correspondence to: Geoffrey P. Margison, Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK. E-mail: gmargison{at}picr.man.ac.uk
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Abstract |
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Abstract
IntroductionIntroduction: Alkylating agents are frequently used in the chemotherapy of many types of cancer. This group of drugs mediates cell death by damaging DNA and therefore, understandably, cellular DNA repair mechanisms can influence both their antitumour efficacy and their dose-limiting toxicities.
Sources of data: This review focuses on the mechanism of action of the DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT) and its exploitation in cancer therapy and reviews the current literature.
Areas of agreement: MGMT can provide resistance to alkylating agents by DNA damage reversal. Inhibition of tumour MGMT by pseudosubstrates to overcome tumour resistance is under clinical evaluation. In addition, MGMT overexpression in haematopoietic stem cells has been shown in animal models to protect normal cells against the myelosuppressive effects of chemotherapy: this strategy has also entered clinical trials.
Areas of controversy: MGMT inhibitors enhance the myelotoxic effect of O6-alkylating drugs and therefore reduce the maximum-tolerated dose of these agents. Retroviral vectors used for chemoprotective gene therapy are associated with insertional mutagenesis and leukaemia development.
Growing points: The results of ongoing preclinical and clinical research involving various aspects of MGMT modulation should provide new prospects for the treatment of glioma, melanoma and other cancer types.
Areas timely for developing research: Tissue- and tumour-specific approaches to the modulation of MGMT together with other DNA repair functions and in combination with immuno- or radiotherapy are promising strategies to improve alkylating agent therapy.
Keywords: AGT • chemotherapy • DNA repair • drug resistance • Lomeguatrib • myeloprotective gene therapy • glioma • MGMT/O6-methylguanine-DNA methyltransferase • O6-benzylguanine • O6-(4-bromothenyl)guanine
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Introduction |
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Treatment for many cancer types is progressing, but for specific disease conditions such as high-grade glioma and metastatic melanoma there is enormous room for improvement. Alkylating agents are important chemotherapeutic drugs for these cancers, lymphoma and others. A substantial amount of preclinical in vitro and in vivo data has demonstrated that the principal mechanism of cell killing by these agents is initiated by O6-alkylguanine formation in DNA and the principal mechanism of resistance to these lesions is mediated by the MGMT DNA repair pathway (see Table 1 for glossary of terms). Considerable effort is currently being directed at improving the efficacy of alkylating agent therapies and the approaches we review here focus on the modulation of MGMT activity to overcome tumour resistance and to ameliorate dose-limiting collateral toxicity.
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Chemotherapeutic alkylating agents and their mechanisms of action |
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The term ‘alkylating agents’ is widely used for any cancer chemotherapeutic drug whose mechanism of action involves attachment of an alkyl group to DNA. This review deals with just one such class of agent, increasingly referred to as ‘O6-alkylating’ drugs, based on the premise that their effects are mediated by reacting with DNA at the O6-position of guanine. O6-Alkylating agents include temozolomide (TMZ), streptozotocin, procarbazine and dacarbazine, which methylate DNA, and BCNU, CCNU and fotemustine, which chloroethylate DNA. Table 2 displays the cancer types that are treated with these agents.
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The mechanism of cell killing by O6-methylguanine (O6-meG) and O6-chloroethylguanine (O6-ClethG) is substantially different, but, in both, DNA replication plays an essential part (Fig. 1). O6-meG frequently mispairs with thymine instead of cytosine during DNA replication. A further round of DNA replication would give rise to the classical G:C to A:T transition mutations that constitute the molecular basis of the mutagenic and carcinogeneic effects of these agents. However, the resulting O6-meG:T mispair can also be recognized by the post-replication mismatch repair (MMR) system, which removes a section of the daughter strand along with the thymine, leaving the O6-meG to again pair with thymine during gap filling. If replication of the gapped structure occurs, doublestrand breaks can be formed which, unless repaired by the recombination repair pathways, result in cell death. Since the toxicity of O6-meG is replication-dependant, methylating agents are only marginally toxic in quiescent cells.
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O6-ClethG, on the other hand, spontaneously converts by internal cyclization into N1-O6-ethanoguanine that then reacts with cytosine on the complementary strand to form a covalent DNA interstrand cross-link. Replication of DNA containing such structures results in stalled replication forks and is potentially lethal. The important distinction is that, for cell killing, methylating agents require a functional MMR system, whereas chloroethylating agents do not.
It should be noted that although O6-alkyguanine is the principal factor in alkylating-agent-induced toxicity, it constitutes only 6–10% of the total damage that is generated in DNA. Varying amounts of 11 other lesions are produced at the various oxygen and nitrogen atoms in DNA bases, in addition to modifications of the oxygen atoms on the phosphate groups. Although the mechanisms of repair of most of these lesions are established, the extent to which they contribute to therapy is still under investigation. Alkylating agents also damage other cellular macromolecules, including RNA, proteins, lipids and low molecular weight components, but there is very little in the literature about the biological effects of these reactions.
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Mechanisms of resistance to O6-alkylating agent chemotherapy |
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Although the alkylating agents are important drugs in cancer chemotherapy, their effectiveness is strongly influenced by DNA repair mechanisms. For both methylating and chloroethylating agents, the initial O6-alkylguanine damage in DNA can be eliminated, and thus toxicity prevented, by the DNA repair protein, MGMT. As mentioned above, for methylating agents, toxicity is mediated by MMR, and downregulation or inactivating mutations of MMR causes resistance (Fig. 1). For chloroethylating agents, DNA repair factors, including Fanconi's anaemia proteins, nucleotide excision repair and homologous recombination seem to be involved in the downstream repair of interstrand cross-links and contribute to resistance (reviewed in Drablos et al.3). Downstream of DNA repair, alkylating agent resistance can also be a consequence of the dysfunctional apoptosis pathways (reviewed in Roos and Kaina5).
Furthermore, there is some evidence that the alkylating agent itself can cause acquired resistance by enriching for MGMT-proficient and MMR-deficient tumour cells or by causing inactivating mutations in MMR genes.6
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O6-Methylguanine-DNA methyltransferase |
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Occurrence and physiological effects O6-Alkylguanine-DNA alkyltransferase was first isolated from E. coli in the late 1970s as the Ada protein, a dual function inducible repair protein, which regulates the adaptive response to low levels of alkylating agents.7–11 E. coli has a second alkyltransferase, the constitutively expressed Ogt protein.12 The human MGMT cDNA was first isolated from a cDNA library by rescue of the ada phenotype in E. coli.13 The characterization of MGMT and its homologues is an ongoing process.
O6-Alkylguanine-DNA alkyltransferases are found in prokaryotes, archea and many eukaryotes, but not in the plant kingdom. The evolutionary conservation of MGMT suggests that it plays a fundamental role in maintaining genomic integrity. However, MGMT is not essential for cell viability. It has been shown for many cell types that sensitivity to alkylating agents inversely correlates with MGMT activity and that MGMT-deficient cells are more susceptible to spontaneous and alkylating-agent-induced toxicity and mutation. Furthermore, MGMT knockout mice are more susceptible to toxicity and tumour induction by alkylating agents, whereas mice overexpressing MGMT are more resistant (reviewed in Margison and Santibanez-Koref4). Thus MGMT protects both normal cells and tumour cells against the toxic and mutagenic effects of O6-alkylating agents and is therefore a crucial factor in mediating the resistance to this class of chemotherapeutic agents.
Mechanism of action MGMT removes the alkyl group from O6-alkylguanine and to a lesser extent from the minor alkylation product O4-alkylthymine in DNA (Fig. 1). Although the methyl group is the preferred substrate, MGMT is also able to remove longer and more complex alkyl groups such as ethyl-, propyl-, butyl-, benzyl- and 2-chloroethyl groups, hence the use of the term ‘alkyltransferase’. Alkyl group removal by MGMT involves the cysteine residue in its active site (Cys145 in the human protein) to which the alkyl group becomes covalently attached. This reaction is stoichiometric and results in the inactivation of the protein, which is subsequently ubiquitinated and degraded by proteasomes. Continued DNA repair requires de novo synthesis of MGMT. Unlike other DNA repair systems, MGMT acts as a single protein and no other enzymes or cofactors are reported to be involved.1–4
The chloroethylation secondary product, N1-O6-ethanoguanine (Fig. 1) is also a substrate for MGMT but in this case the protein becomes covalently bound to the N1 position of guanine via an etheno link; this has, to date, no known biological significance.
The transfer of the alkyl group to MGMT leads to a change in protein conformation and the exposure of a specific motif (LXXLL). One report suggests that this motive binds to the oestrogen receptor (OR) and that alkylated MGMT thus blocks transcription coactivator binding to OR, resulting in the inhibition of transcription.14 In this study, following repair of alkylation damage in DNA, MGMT arrested OR-mediated cell proliferation. The dual function of MGMT as a DNA repair protein and inhibitor of OR-mediated cell proliferation may have implications in carcinogenesis and progression of oestrogen-associated tumours such as breast and endometrial cancer. However, in a more recent study no interactions of OR and MGMT were seen and therefore the physiological relevance of such a mechanism is questionable.15
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Factors influencing MGMT expression |
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Although MGMT is ubiquitously expressed, its activity varies considerably between human tissues: the liver expressing the highest levels and haematopoietic tissues and brain the lowest. There is also a wide range of interindividual variation in MGMT expression. The factors that influence these levels of MGMT are not fully defined, but they are expected to impact on the sensitivity of normal tissues to the side effects of chemotherapeutic alkylating agents and are strongly suspected to influence susceptibility to cancer. Tumour tissues express very different amounts of MGMT, from levels much higher than normal tissues, to undetectable levels in a portion of some tumour types (see below).
Transcription factors and p53 The MGMT promoter sequence contains several transcription-factor-binding sites including glucocorticoid response element and activator protein-1 sites.16 Induction via these sites in vitro results in moderate increases in MGMT expression that are sufficient to increase resistance to alkylating agents (reviewed in Sabharwal and Middleton2). The synthetic glucocorticoid, dexamethasone is routinely used in glioma to reduce the effects of oedema. Since dexamethasone has been shown to upregulate MGMT in vitro, it might reduce the effectiveness of alkylating agents in glioma therapy.17
In rodents, DNA damage has been shown to induce MGMT expression, and there is some evidence that this may occur in human cells and tissues. Ionizing radiation upregulates MGMT expression in mice but not in knockout mice deleted for the tumour suppressor, p53.4,18 A recent study showed that p53 directly induces MGMT expression in murine astrocytic glioma cells by binding to the promoter region.19 In agreement with this, the inactivation of p53 has been reported previously to sensitize some astrocytoma cell lines to TMZ and BCNU. This is in apparent contrast to a study showing that the overexpression of p53 in vitro suppresses transcription of MGMT in human fibroblast cell lines.20
Promoter methylation Reduced MGMT activity in cultured tumour cells and human tumours is often the result of epigenetic silencing by promoter methylation, (in this case mediated physiologically by DNA-cytosine methyltransferase, not methylating agents). Methylation of the CpG islands in the MGMT promoter region leads to the formation of inactive chromatin that limits transcription. Patients who were newly diagnosed with glioblastoma and who had methylated MGMT promoter (45% of the cohort) had a significant survival benefit following the treatment with TMZ and radiotherapy compared with those who were assigned to only radiotherapy. Similarly, the analysis of gliomas obtained from BCNU-treated patients showed that MGMT promoter methylation (observed in 40% of the samples) was associated with regression of the tumour and prolonged overall survival. Promoter hypermethylation in 36% of patients with diffuse large B-cell lymphoma resulted in significant increase in overall survival of patients treated with multidrug regimens including the alkylating agent, cyclophosphamide.2,21 Therefore, MGMT promoter methylation status is emerging as a prognostic factor for tumour therapy and is currently being assessed for selecting glioma patients most likely to benefit from alkylating drug treatment. However, contradictory results were obtained using other methods to assess MGMT expression in gliomas, i.e., immunohistochemistry or activity determination. The heterogeneity of glioma biopsies is an additional complicating factor. Although promoter methylation is clearly involved in gene silencing of MGMT, other factors may participate. There is also the possibility that MGMT promoter methylation is a marker for general epigenetic deregulation and that genes other than MGMT are more relevant to clinical outcome.
In addition, in vitro studies indicate that the methylation of CpG sites in the body of the gene are important in MGMT expression, and this suggests that methylation analysis in any clinical context should be extended to the coding regions of the gene.22
Single nucleotide polymorphisms Interindividual variation in MGMT can be influenced by intragenic single nucleotide polymorphisms (SNP) (reviewed in Bugni et al.15). One such SNP, Ile143Val, affects an amino acid close to the Cys145 residue at the active site of MGMT. This does not affect the stability or activity of the protein on methylated DNA substrate, but the protein has been shown to be more resistant to inactivation by MGMT pseudosubstrates (see below).23 Women homozygous for the Val143Val variant showed a significantly increased risk for cervical carcinoma.24 Additional studies are required to reveal the mechanisms by which MGMT variants might influence the response of normal and tumour tissues to both the toxic and mutagenic effects of O6-alkylating agents.
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Strategies to improve tumour killing by O6-alkylating agents |
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Several strategies are being pursued to improve tumour cell killing by O6-alkylating agents. These include attempts to increase tumour cell sensitivity by depletion of MGMT by the alkylating drug itself, by pseudosubstrates, or by RNA interference (RNAi) and viral proteins.
Alkylating agent combinations O6-Alkylating drugs deplete MGMT activity indirectly via alkylation of DNA. Several studies have exploited the ability of methylating agents to reduce MGMT activity prior to administration of chloroethylating agents. Although these combinations showed MGMT depletion, they also resulted in significant additional toxicity, mainly increased myelosuppression, but also acute lung toxicity, so far without improved tumour responses. Further trials of this strategy are ongoing.
Inactivation of MGMT by low molecular weight pseudosubstrates An increasingly popular approach to enhance efficacy is to inactivate MGMT by means of direct inhibitors prior to O6-alkylating agent therapy (reviewed in Rabik et al.25). Several analogues of O6-meG have been synthesized (Fig. 2). They act as pseudosubstrates for MGMT and alkyl group transfer results in the inactivation of MGMT.
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O6-Benzylguanine (O6-BG) is so far the most extensively studied direct MGMT inhibitor (Fig. 2C) (reviewed in Rabik et al.). In a variety of human tumour cell lines and xenograft models including melanoma, brain, prostate and colon cancer, O6-BG was shown to inactivate MGMT and to enhance tumour growth inhibition in combination with TMZ or BCNU. O6-BG has been shown to readily pass the blood–brain barrier and has therefore potential for the treatment of brain tumours. Several clinical trials of the combination of O6-BG and BCNU or TMZ are now complete (Phases I and II) or still ongoing (Phases II and III) in brain tumours, melanoma, lymphoma, colon cancer or sarcoma. In Phase I studies, O6-BG at a dose of 120 mg/m2, which was inherently non-toxic, completely inactivated tumour MGMT activity. However, at this dose, O6-BG increased the myelosuppressive effects of BCNU, and the maximum-tolerated dose (MTD) of BCNU was approximately 3-fold lower than the MTD for BCNU alone.
Another pseudosubstrate, O6-(4-bromothenyl)guanine [O6-BTG, patrin, Lomeguatrib (LM)] (Fig. 2D) was reported to be more potent than O6-BG in inactivating recombinant MGMT protein and is orally bioavailable. In human tumour cell lines, primary cells and xenografts such as ovarian cancer, acute leukaemia, melanoma and breast tumour, LM efficiently and rapidly inactivated MGMT, was not inherently toxic and significantly increased the tumour growth inhibitory effect of TMZ.26–29 In combination with BCNU, LM was less toxic than O6-BG but resulted in similar tumour growth delay. A Phase I trial of LM in combination with TMZ in advanced solid tumours determined a regimen of oral LM 40 mg/day and TMZ 125 mg/m2 on days 1–5.30 This reduction, from a normal dose of 200 mg/m2, was necessary to bring the LM-mediated increased bone marrow toxicity back to an acceptable level. LM is currently undergoing Phase II clinical trials in metastatic malignant melanoma.
It seems likely that the optimal exploitation of direct MGMT inactivators will require strategies to circumvent the increased collateral toxicity of the O6-alkylating drug. This might be achieved by targeting the alkylating agent or inactivator specifically to tumour cells or by increasing the resistance of the bone marrow, which is the most susceptible normal tissue, by means of myeloprotective gene therapy (see below).
Inactivation of MGMT by RNAi and viral proteins RNAi technology can be used to silence DNA repair genes and can render a variety of cancer cell lines sensitive to chemotherapeutic agents in vitro (reviewed in Pai et al.31). Thus, transient transfection of human nasopharyngeal carcinoma cells with MGMT-targeted short-interfering RNA (siRNA) causes a 4-fold increased sensitivity to BCNU in vitro.32 However, a major challenge of RNAi technology is in vivo delivery (reviewed in Snove and Rossi33). Viral vectors can be used for transient or persistent expression of siRNA in the form of short hairpins (shRNA) in vivo. Commonly used vectors include the nonintegrating adenovirus, adeno-associated virus and herpes simplex virus and the integrating oncoretroviruses and lentiviruses. The vector delivery can be either local or systemic. Pseudotyping of viral vectors or tissue-specific expression by appropriate polymerase II promoters or even inducible expression, for example by the tetracycline systems are in development to allow targeted and controlled expression of shRNAs in vivo.
It has been reported that the E1A gene product of adenovirus efficiently inhibits the promoter activity of MGMT and may thus reduce chemoresistance. Therefore, gene therapy vectors based on replication competent adenoviruses could combine the features of an oncolytic virus with the possibility of potentiating chemotherapy by inhibiting DNA repair mechanisms. Phase I/II clinical studies with the E1B mutant oncolytic adenovirus ONYX-015 and the DNA-damaging agent cisplatin have shown a favourable effect of combined therapy and are relatively safe (reviewed in Jiang et al.34). In China, after a successful Phase III clinical trial, virotherapy using the E1B mutant adenovirus H101, which is similar to ONYX-015 was approved in 2005 for use in combination with cisplatin and 5-fluorouracil for patients with head and neck cancer. This world-first oncolytic viral therapy has been a great step for cancer therapy, although the approval has been a controversial subject within the scientific community (reviewed in Garber35).
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Strategies to reduce the toxic side effects of O6-alkylating agents |
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Myelosuppression is the principal dose-limiting toxicity when using O6-alkylating drugs as single agents. As outlined above, when combined with MGMT inactivators, this is substantially increased, requiring considerable drug dose reductions. Local drug administration that reduces the levels of damage introduced into bone marrow, or gene therapy regimens that protect bone marrow cells would allow intensification of therapy and hence increased tumour responses.
Furthermore, the role of alkylating agents in the development of secondary acute leukaemia is well recognized. TMZ has mutagenic potential as shown in murine models,36,37 (reviewed in Margison and Santibanez-Koref4). Myeloid malignancies have been reported in clinical studies shortly after combination treatment including TMZ, suggesting the promotion of secondary tumour development by TMZ.38,39 Local drug administration and myeloprotective gene therapy would also substantially reduce the risk of therapy-related neoplasia development.
Local drug administration Attempts have been made to reduce systemic toxicity by local drug administration. A rat model of extremity melanoma was treated by isolated limb infusion with high-dose TMZ and O6-BG. This regimen showed a marked reduction in tumour growth with minimal systemic toxicity.40
In patients with recurrent malignant glioma, BCNU in wafer form (Gliadel) was inserted directly into the affected area after resection of the tumour.41 The patients then received, intravenously, 120 mg/m2 of O6-BG followed by a continuous infusion of 30 mg/m2/day for up to 2 weeks. This dosing resulted in undetectable MGMT levels in tumour tissue and no additional toxicity was observed. A Phase II study of Gliadel with O6-BG is ongoing in recurrent glioblastoma multiforme, in order to define the antitumour efficacy of this regimen.42
In an alternative approach to attenuating the increased systemic toxicity caused by MGMT inactivation, by-weekly intracerebral O6-BG was administered using an Ommaya reservoir in conjunction with systemic TMZ therapy.43 The local administration of O6-BG was well tolerated without any complications. MGMT activity in the residual tumour was not determined and thus the level of depletion can only be speculated. Whether this strategy will indeed result in increased therapeutic response will be established in future trials.
Myeloprotective gene therapy The myelosuppressive effects of O6-alkylating agents are likely due to the low levels of expression of MGMT in bone marrow. In support of this, it has been shown that MGMT levels in peripheral blood mononuclear cells, which probably reflect those in bone marrow stem cells, correlate with myelotoxicity in TMZ dose escalation studies.44 Considerable effort has been directed towards augmenting MGMT activity by myeloprotective gene therapy (reviewed in Zaboikin et al.45). To achieve this, MGMT-expressing retrovirus or lentivirus has been used for ex vivo transduction of murine bone marrow. To combine this approach with MGMT inactivation in tumour, mutant versions of MGMT (G156A or P140K), which are resistant to inactivation by O6-BG or LM, have been used.46 Following transplant of the gene modified cells into recipients, high level expression of MGMT has been seen in bone marrow and peripheral blood cells. Several groups have shown that this strategy provides protection of haematopoietic stem cells against MGMT inactivator-O6-alkylating agent combinations in vitro and in vivo in murine47,48 and canine models49 and has enabled increased tumour kill in mice.50,51
Currently, three Phase I clinical trials of this myeloprotective gene therapy strategy are ongoing in the US.52 In two of these trials, patients with solid tumours or non-Hodgkin's lymphoma are receiving autologous haematopoietic stem cells. These have been harvested from peripheral blood and transduced with a retrovirus harbouring an inactivator-resistant MGMT. Patients are being treated with O6-BG to overcome tumour resistance, in conjunction with TMZ or BCNU. Such studies will evaluate the degree of cell engraftment, the feasibility of in vivo enrichment of the transduced haematopoietic progenitors, the durability of transgene expression and the toxicity of the chemotherapy regimen. The third study with brain tumour patients will investigate the outcome of an intensified combination chemotherapy with lomustine, vincristine and procarbazine along with peripheral blood stem cell retroviral transduction of MGMT.
The outcome of these clinical trials will provide important information on how effective this approach will be in simultaneously increasing the chemosensitivity of tumours and protecting blood progenitor cells against the toxic effects of drug-induced damage. Having provided protection in the bone marrow compartment, acute toxicity in other tissues might be anticipated, especially if dose escalation is achieved. In the long-term survivors of this therapy, it will also be interesting to observe if there is protection against the myelodysplasia and leukaemogenic effects of the O6-alkylating agents.
Some concern has been expressed about the use of retrovirus and the associated risk of insertional mutagenesis.53,54 However, the prognosis for patients that would be treated with this therapy is very poor, and the risk is considered worthwhile especially since recent modifications in vector design are reducing the risk of insertional mutagenesis (reviewed in Schambach and Baum55). There is also some indication that the long-term overexpression of mutant MGMT in haematopoetic stem cells may be a cell growth disadvantage. It is not known if this will be a problem in patients, but vectors for inducible or low level expression of MGMT are currently in preclinical development.
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Conclusion |
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The therapeutic outcome of O6-alkylating agent chemotherapy is largely affected by the action of the DNA repair protein MGMT, high levels of which in tumour cells mediates tumour chemoresistance. Identification of patients suitable for O6-alkylating agent chemotherapy by assessing tumour MGMT expression, or inhibition of tumour MGMT to overcome resistance, is a reasonable strategy to improve the therapeutic index of these agents.
Low MGMT levels of normal cells limit the doses used in O6-alkylating agent chemotherapy. Gene therapeutic augmentation of bone marrow MGMT to attenuate sensitivity has potential to reduce the systemic toxicity of these types of chemotherapeutic agents and to allow drug dose escalation. An additional application of this strategy is the selection of gene modified stem cells for correction of monogenetic disorders.56
Recent approaches to increase tumour sensitivity to O6-alkylating agents have involved targeting base excision repair and other DNA repair pathways (reviewed in Madhusudan and Middleton21). Combinations of DNA-repair-targeting strategies with immunotherapy or radiotherapy are promising to achieve further tumour reduction and an overall survival benefit for cancer patients receiving O6-alkylating agent chemotherapy. For example, in a clinical pilot study, IL-2 immunotherapy to favour recovery of immunological reactivity has been combined with LM plus TMZ chemotherapy in refractory acute leukaemia patients.57
Given these developments, the present poor prognosis, particularly for melanoma and glioma patients, is surely set to improve.
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Funding |
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The authors acknowledge the support of Cancer Research UK. DEG acknowledges the support of EU-ATTACK FP6 programme (LSHC-CT-2005-018914).
Accepted for publication December 14, 2007.
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