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Thiopurines in current medical practice: molecular mechanisms and contributions to therapy-related cancer
Peter Karran and Natalie Attard

Abstract | Thiopurines have diverse clinical applications and their long-term use as antirejection drugs in transplant patients has been associated with a significantly increased risk of various types of cancer. Although they are slowly being replaced by a new generation of non-thiopurine immunosuppressants, it is anticipated that their use in the management of inflammatory and autoimmune diseases will continue to increase. Therapy-related cancer will remain a potential consequence of prolonged treatment for these generally non-life-threatening conditions. Understanding how thiopurines contribute to the development of cancer will facilitate clinical decisions about the potential risks to patients of long-term treatment for chronic inflammatory disorders.
The thiopurines azathioprine, 6-thioguanine (6-TG) and 6-mercaptopurine (6-MP) are effective antiinflammatory, anticancer and immunosuppressive drugs. They have been available in clinical practice for over half a century, with 6-MP and azathioprine receiving US Food and Drug Administration (FDA) approval in 1953 and 1968, respectively1. The arena for thiopurine medication has significantly diversified since their introduction and both the on- and the offlabel applications for inflammatory and autoimmune diseases are extensive. The International Agency for Research on Cancer (IARC) has, however, determined that there are sufficient grounds to classify azathioprine as a human carcinogen2, and it is now well recognized that prolonged treatment with thiopurines is associated with an increased risk of various malignancies. In support of this conclusion, IARC cites the increased incidence of non-Hodgkin lymphoma, skin squamous cell carcinoma (SCC), hepatobiliary carcinoma and mesenchymal tumours in renal transplant patients. The IARC report also notes that the same malignancies are more frequent albeit to a lesser extent in patients treated with azathioprine for, inter alia, rheumatoid arthritis, systemic lupus and inflammatory bowel disease (see section on chronic inflammatory disorders). Much of the increased cancer in transplant patients can be attributed to oncogenic viruses, probably as a consequence of immunosuppression, and is not directly related to the mechanism of action of azathioprine. The important contribution of immunosuppression is further highlighted by the parallels between the range of cancers in transplant recipients and among HIVinfected individuals3,4. Kaposi sarcoma, non-Hodgkin lymphoma, liver cancer and cervical cancer are frequent in both groups. Among transplant patients, the post-transplant lymphoproliferative disorders, which include non-Hodgkin lymphoma, are also likely to be of viral origin. These malignancies, which are second only to skin SCC in frequency, are predominantly early-onset and occur mostly within the first year after transplant. A possible viral aetiology for SCC in transplant patients has not been excluded, although evidence for a causative association with known oncogenic human papillomavirus types has proved elusive5. It remains possible therefore, that some of the cancer associated with azathioprine treatment reflects the properties of 6-TG incorporated into DNA. DNA 6-TG can be considered the ultimate active metabolite of all the thiopurines. 6-TG accumulation in DNA is an inevitable consequence of thiopurine treatment and might be central to their therapeutic effects. We describe some of the chemical properties of DNA 6-TG and explore, in broad terms, how these might accelerate carcinogenesis. As clinical examples, we will consider myelodysplastic syndrome-associated acute myeloid leukaemia (AML) and skin cancer in organ-transplant recipients. We also address the potential consequences of long-term thiopurine treatment for chronic inflammatory conditions.
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Cancer Research UK London Research Institute, Clare Hall Laboratories, South Mimms, Hertfordshire, EN6 3LD, UK. Correspondace to P. K. e-mail: Peter.Karran@cancer.org.uk doi:10.1038/nrc2292

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At a glance
The thiopurines azathioprine, 6-mercaptopurine and 6-thioguanine (6-TG) have been available to medical practitioners for over half a century. They are used as anticancer and immunosuppressive agents. The introduction of azathioprine as an immunosuppressant revolutionized solid-organ transplantation from unrelated donors and resulted in much improved graft survival. The thiopurines are recognized treatment options for an increasing number of chronic inflammatory and autoimmune disorders, including arthritis and colitis. Largely on the basis of epidemiological data of cancer in transplant patients, the International Agency for Research on Cancer classifies azathioprine as a human carcinogen. Much of this increased cancer can be attributed to the effects of immunosuppression and the involvement of oncogenic viruses. In some cases, however, demonstration of a viral aetiology has proved elusive. This is particularly true of skin cancer, which is the major treatment-related cancer among transplant patients. Thiopurines are prodrugs and one outcome of their complex metabolism is the incorporation of 6-TG into DNA during replication. 6-TG is chemically more reactive than canonical DNA bases and undergoes methylation insituin DNA. Methylated DNA 6-TG is ultimately cytotoxic by a mechanism that depends on the cells DNA mismatch repair system. One route of escape from the cytotoxicity of thiopurines is by inactivation of mismatch repair. Mismatch repair defects are associated with high rates of spontaneous mutation and are common in certain types of cancer. Acute myeloid leukaemia occurs more frequently than expected in transplant patients. These azathioprine-related cancers are often defective in mismatch repair. DNA 6-TG is also photochemically reactive and has a maximum absorbance at 340 nm in the UVA region of the ultraviolet spectrum. UVA comprises more than 90% of solar radiation that reaches the earth and, on exposure to UVA, the 6-TG DNA chromophore generates reactive oxygen species (ROS), which can damage DNA, proteins and other cellular macromolecules. DNA 6-TG itself is particularly susceptible to oxidation by ROS to form guanine-6-sulphonate. This photoproduct is a powerful block to replication but can be bypassed by Y-family polymerases which have a relatively relaxed stringency. The photochemical reactions of DNA 6-TG are mutagenic and this might contribute to an increased risk of transplantrelated squamous cell carcinoma of the skin. The association of azathioprine with therapy-related cancers and its increasing use in treatment of chronic inflammatory and autoimmune disorders suggests that careful monitoring of these patients for signs of possible therapy-related cancer is advisable.

Therapeutic indications for thiopurine treatment The principal therapeutic effects of thiopurines can be divided into two broad clinical categories: anticancer and immunosuppressive. Their pharmacological mode of action will be described in detail in the next section.
Anticancer. Initially developed as an antagonist of purine utilization, 6-MP was shown to inhibit tumour growth in rodents6. The pioneering use of 6-MP as a treatment for acute lymphoblastic leukaemia7, the commonest childhood cancer, contributed to a dramatic improvement in survival from this malignancy, which previously had a dismal prognosis. Immunosuppression. Shortly after its development, 6-MP was shown to have immunosuppressive properties. one of the outstanding successes of the thiopurines, principally azathioprine, since their introduction in the 1960s is their contribution to increased graft survival following organ transplant. In combination with corticosteroids, they dominated this field for nearly two decades. Azathioprine is increasingly being replaced in this context by the newer generation of immunosuppressants such as mycophenolate mofetil, tacrolimus and rapamycin (see FIG. 1a) and the extent to which azathioprine has been phased out as an immunosuppressant in renal transplant patients over the past decade is shown in FIG. 1b. In particular, by 2003 mycophenolate mofetil a prodrug of mycophenolic acid, which is an inhibitor of inosine monophosphate
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dehydrogenase (see the following section) was prescribed for approximately 80% of renal transplant patients in the United States at the time of discharge from hospital. The wisdom of the worldwide replacement of azathioprine by mycophenolate mofetil is brought into question by a recent study in which renal transplant patients receiving the newer microemulsion ciclosporin preparation in combination with either azathioprine or mycophenolate mofetil achieved comparable long-term outcomes in terms of graft survival8. In addition, the average cost per patient is 15-fold higher for mycophenolate mofetil than for azathioprine, raising questions about the cost-effectiveness of patient care9. In view of their relatively recent introduction into routine clinical practice, whether these novel immunosuppressants contribute to post-transplant malignancy remains an open question10. The immunosuppressive properties of thiopurines are exploited in the management of a number of chronic inflammatory and autoimmune diseases, including rheumatoid arthritis, systemic lupus erythematosus, pemphigus vulgaris, dermatomyositis, multiple sclerosis and myasthenia gravis. In this category, probably the most long-standing experience has been gained in inflammatory bowel disease (IBD) for which thiopurines have been used since the 1960s11. Thiopurine use remains widespread and, in effect, doctors in most hospital-based medical specialities will look after patients who are taking or have taken thiopurine drugs at some point. Their acute life-threatening side effects are well known12. It is equally important
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a
Calcineurin inhibitors: Ciclosporin Tacrolimus Disrupt cytokine gene expression by NFAT inhibition mTOR inhibitors: Rapamycin (sirolimus) Everolimus Inhibit serine/threonine protein kinase activity of mTOR

Antimetabolite: Aza

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Figure 1 | Immunosuppressant therapy in transplant patients. a | The principal immunosuppressant drugs used in solid organ transplants, when they were introduced into routine clinical practice and the principal mechanism| of Nature Reviews Cancer action of the newer drugs are indicated in the timeline. b | Trends in immunosuppressant use. (Over the past decade novel immunosuppressants, including the mammalian target of rapamycin (mTOR) inhibitors and mycophenolic acid, have been prescribed more frequently.) The bars in the figure represent the number of patients in the United States treated with a particular immunosuppressant at discharge from hospital. By 2003, the proportion of new transplant patients who were taking azathioprine (aza) had declined to <5%. Data shown here are for kidney transplants. The same trend is apparent for heart transplants with ~10% of new recipients starting immunosuppression with aza in 2003. Data taken from the United States Organ Procurement and Transplantation Network and The Scientific Registry of Transplant Recipients 2004 Annual Report. IMPDH, inosine monophosphate dehydrogenase; MMF, mycophenolate mofetil; NFAT, nuclear factor of activated T cells.

Year

Year

that the potential carcinogenic consequences of their prolonged use are also appreciated. Understanding how these drugs are carcinogenic could result in these distressing side effects being minimized or eliminated.

Purine salvage pathway

Cells can obtain the purine bases they need to form the precursors of DNA and RNA either by synthesizing them denovoor by recycling from degraded nucleic acids through this pathway.

Myelosuppression
A condition in which the production of blood cells by the bone marrow is significantly reduced. This can result in anaemia, lifethreatening infection and spontaneous bleeding.

Metabolism, immunosuppression and cytotoxicity Thiopurines are prodrugs and need metabolic conversion into active compounds13, although the contribution of various active metabolites to their pharmacological effects has not been unequivocally defined. The first step of azathioprine activation is the removal of the substituted imidazole ring in a non-enzymatic reaction involving glutathione. This occurs in erythrocytes which then release 6-MP, the active metabolite. As with 6-TG, 6-MP is readily transported into cells14, where it enters the purine salvage pathway. Both thiopurines are good substrates for hypoxanthineguanine phosphoribosyltransferase 1 (HPRT1), the first enzymatic step of salvage. HPRT catalyses the addition of ribose 5-phosphate to generate thioinosine monophosphate (TIMP) and thioguanosine monophosphate (TGMP) from 6-MP and 6-TG, respectively (FIG. 2). This is the first step in their conversion to thioguanine nucleotides (TGNs), which are precursors for the incorporation of 6-TG into RNA and DNA. In competing reactions, the degradation of intracellular 6-MP is initiated by xanthine oxidase and both 6-MP and 6-TG are inactivated by thiopurine S-methyltransferase (TPMT)15.

The normal function of human TPMT, which is conserved among species from bacteria to mammals16, is unknown. The bacterial homologues convert selenium- and tellurium-containing compounds to less toxic methylated derivatives using S-adenosylmethionine (SAM) the source of methyl groups for numerous enzyme reactions as a methyl group donor17. There is speculation that the human enzyme might also detoxify these metabolically important, but potentially poisonous, metals16. The best characterized activity of human TPMT is, however, the detoxification of 6-MP and 6-TG which, together with TIMP and TGMP18, are converted to inactive methylated products. Methylation of all these intermediates impairs the synthesis of TGNs and thereby reduces the effective thiopurine dose. The significance of this catabolism is emphasized by the observation that the <1% of patients who inherit two low-activity variant alleles of the TPMT gene suffer intense myelosuppression and huge decreases in whole-blood cell counts. This is a potentially fatal reaction to what is effectively a thiopurine overdose and pretreatment screening for low TPMT is now routine in some institutions19 (BOX 1).

Mechanisms of thiopurine toxicity DNA mismatch repair. As noted above, the metabolism of 6-TG and 6-MP culminates in the formation of 6-thiodGTP (FIG. 2), which is a good substrate for replicative DNA polymerases20,21 with a reported apparent
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O2N S HN N N N N N H CH3 HN H2N N S N N H

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S HN N N N O OH OH HN HO N O OH OH S N N HN H 2N N O OH OH S N N HN S N N O OH N

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Figure 2 | Thiopurines and their metabolism. Azathioprine is converted to 6-mercaptopurine (6-MP) by non-enzymatic activation in red blood cells. Both 6-MP and 6-thioguanine (6-TG) are salvaged by the hypoxanthineguanine phosphoribosyltransferase (HPRT) of immune effector cells and converted into their respective nucleoside monophosphates (TIMP and TGMP). In competing catabolic reactions, thiopurine S-methyltransferase (TPMT) inactivates 6-MP and 6-TG by S-methylation and xanthine oxidase (XO) converts 6-MP to 6-thiouric acid. TIMP and TGMP are also TPMT substrates. Methylated TIMP (meTIMP), but not meTGMP, is an effective inhibitor of denovo purine biosynthesis. TIMP that escapes catabolism is further metabolizedbyinosine monophosphate dehydrogenase (IMPDH) and guanine monophosphate synthetase (GMPS) to TGMP. Sequential action of deoxynucleoside kinases and reductase generates the thioGTP and thio-dGTP that are the substrates for incorporation of 6-TG into RNA and DNA.

Nature Reviews | Cancer

Km

Derived from enzyme kinetics, Km is the substrate concentration at which an enzymatic reaction proceeds at half-maximal velocity. It is effectively a measure of the affinity of an enzyme for a particular substrate.

similar to that of unmodified dGTP. 6-TG is not known to be subject to prereplicative excision repair and can accumulate in DNA. Replication of a 6-TG in the template DNA strand is also relatively easy and the thiopurine is not a major obstacle to elongation by DNA polymerases in simple primer extension assays21,22. Coding by 6-TG is ambiguous, however, and C and T are incorporated with approximately equal facility 23. In intact cells, a low level of DNA substitution by 6-TG is neither toxic nor particularly mutagenic. Higher levels induce significant toxicity that is absolutely dependent on active purine salvage. As cells have only a single active copy of the X-linked HPRT1 gene that encodes the key enzyme in the salvage pathway, they acquire resistance to high concentrations of 6-TG by a single mutational event that inactivates HPRT. This selection for 6-TG resistance is the basis of the widely used HPRT mutation assay in cultured human cells. Thiopurine toxicity is a delayed effect. This partly reflects the requirement for passage through one S phase of the cell cycle to allow incorporation of 6-TG into DNA. In fact, cytotoxicity is delayed even longer than this, suggesting a requirement for passage through
Km

at least one more S phase to allow replication of the 6-TG substituted DNA24,25. This extensive delay, and the involvement of replication of the modified DNA, is an unusual property shared with methylating agents that produce DNA O6-methylguanine (O6-meG)2627 a DNA lesion that is structurally similar to 6-TG. The observation that cells could develop simultaneous resistance, known as methylation tolerance, to methylating agents and to thiopurines suggested that the cytotoxic effects of these two very different classes of drug involve a common mechanism28 related to the similarity between O6-meG and 6-TG. Methylation tolerance was shown to be genetically recessive 29,30, consistent with an escape from cell death by loss or inactivation of a function required to process both DNA 6-TG and DNA O6-meG. earlier experiments in bacteria 31 indicated that the DNA mismatch repair system (MMR) was a likely suspect for processing O6-meG-containing, and by extension, 6-TG-containing, base pairs that share some of the geometrical properties of bona fide mismatches32 (FIG. 3). Processing caused cell death, and toxicity was alleviated by mutations that disabled MMR.
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Box 1 | Thiopurine S-methyltransferase testing in the clinic
The value of thiopurine S-methyltransferase (TPMT) testing before initiation of azathioprine or 6-MP therapy is contentious. Alleles for low- and high-level TPMT activity are inherited in an autosomal co-dominant manner98 resulting in three distinct phenotypes: normal/high, intermediate and deficient S-methylators, comprising approximately 89%, 11% and 0.3% of the population, respectively. The most common variant alleles encoding low TPMT are designated TPMT*3A and TPMT*3C. The 1 in 300 people who inherit a combination of two low-activity TPMTalleles are deficient in thiopurine methylation and suffer severe and early myelotoxicity after taking azathioprine or 6-MP. The clinical rationale for prior testing is to identify these rare instances of TPMT enzyme deficiency and consequently either avoid azathioprine and 6-MP entirely or prescribe at a much lower dose with intensified monitoring for possible toxic side effects. Additional aims are dose modification to reduce toxicity in patients with an intermediate phenotype and to avoid sub-optimal treatment of patients with normal or high enzyme activity. The additional clinical benefit that identifying TPMT status has over traditional monitoring of clinical and serological parameters is not clear, and it is uncertain whether this knowledge can be used to individualize a dosing regimen. Mercaptopurine metabolism is complex and may be influenced by factors such as underlying disease, co-medication and race99. Furthermore, TPMT status is not the only determinant of myelosuppression, which remains a potential consequence even in patients with normal TPMT activity. The need for close monitoring of blood counts is not negated in these patients and published reports of cost-effectiveness of routine TPMT testing are conflicting100,101. Despite this, prior TPMT testing is recommended by the Food and Drugs Administration in the USA and is recognized as best practice in authoritative guidelines in dermatology102. Its usefulness remains unsupported by firm evidence.

To link active MMR to cell death in human cells, a model was proposed28 in which post-replicative processing by MMR of aberrant base pairs containing 6-TG or O6-meG generated potentially lethal DNA lesions because the aberrant base was in the template (FIG. 3b), rather than in the daughter DNA strand, which MMR normally edits (FIG. 3a). Processing of these particular base pairs was inevitably futile because DNA polymerases could never find a sufficiently precise daughter-strand partner for the rogue base that would allow it to escape the attention of MMR. The replication fork is, meanwhile, free to advance leaving these unresolved processing attempts in its wake. Some of the molecular events underlying the delayed cell-cycle effects that are associated with MMR-dependent DNA damage interactions have been defined and these provide support for the futile processing model. Methylating agents or 6-TG provoke a delayed MMRdependent G2 cell-cycle checkpoint25,33,34. Careful analysis reveals that MMR-dependent G2 checkpoint activation is mediated by ATR (ataxia telangiectasia and Rad3related)ATRIP (ATR interacting protein) and CHeK1, which are associated with aberrant events at replication forks34,35. The ATM DNA damage sensor is not required for this DNA damage response but is activated in a MMRindependent manner by DNA methylation damage other than O6-meG36,37. ATR and CHeK1 activation depends on MutS (an MSH2 and MSH6 dimer) and MutL (a dimer of MLH1 and PMS2) and it seems likely that the late triggering of the checkpoint is caused by anomalous DNA structures38 generated during replication through persistent unresolved MMR intermediates from the previous cell cycle. one of the most recent biochemical studies provided direct evidence for the repetitive
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processing of O6-meG:T base pairs by MMR in cell extracts39. Direct association of ATRATRIP with MutS and MutL35, or the recruitment of apoptotic signalling factors at MutS bound to the aberrant base pairs40, have also been proposed as mechanisms by which cellular signalling or apoptosis might be initiated without the need for mismatch correction attempts. overall, the timing of the DNA-damage-checkpoint response supports the futile processing concept and most of the evidence suggests that base pairs containing O6-meG or 6-TG do not directly provoke G2 arrest or cell death. The potentially lethal DNA damage that effects checkpoint activation is caused by replication through regions of unresolved MMR attempts generated during the previous S phase. The cells that undergo ATRCHeK1-related G2 arrest after the second S phase following damage almost certainly contain aberrant DNA structures that have a high probability of being lethal and can be repaired by recombination. Methylating agents and 6-TG are associated with characteristic MMR-dependent recombination events41,42. It follows from this that inactivation of MMR, by preventing the formation of potentially lethal doublestrand breaks, increases cell survival. This tolerance of methylation or 6-TG-related DNA damage comes at the cost of a significantly increased rate of spontaneous mutation due to uncorrected replication errors. The likelihood of a cell acquiring mutations that allow it to escape from the normal constraints on growth is thereby increased and the time required for malignancy to develop is reduced. By way of illustration, inherited mutations in genes encoding components of MutS or MutL cause early-onset cancer in people with hereditary nonpolyposis colorectal cancer syndrome43. Although the original futile processing model provided a satisfying qualitative explanation for the involvement of MMR in the toxicity of methylating agents and thiopurines, there were some quantitative inconsistencies. equitoxic treatments were associated with approximately 1,000 DNA O6-meG lesions but as many as 107 DNA 6TGs. This puzzling discrepancy was resolved by Swann and his colleagues44, who demonstrated that the base pairs that provoke MMR involve a methylated form of DNA 6-TG (me6-TG) that is generated by an in situ, nonenzymatic reaction of DNA 6-TG with SAM, which is a weak methylating agent. only ~1 in 104 DNA 6-TGs undergoes methylation. Thus, when cells are treated with thiopurines or methylating drugs, similar numbers of DNA me6-TG (through SAM methylation) and O6-meG (from direct methylation) lesions cause similar extents of cell killing. SAM-mediated chemical methylation of DNA 6-TG provides a striking example of the increased reactivity that accompanies replacement of the guanine oxygen atom by sulphur45. Methylation of the o6 atom of guanine by SAM is unfavourable and would only generate approximately 10 DNA O6-meGs during a typical S phase46. DNA 6-TG is much more reactive and the rate of S-methylation is nearly two orders of magnitude higher than that of guanine when cells are treated with weak methylating drugs47. This enhanced reactivity means that DNA 6-TG becomes methylated to a significant extent, even at relatively low intracellular concentrations of SAM.
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Polymerase-associated G PCNA sliding clamp 5 T

3 G MutS Recognition and processing 5 T

3 MutL Excision and resynthesis EXO1 PolPCNA 5 G C

DNA polymerase Daughter strand of DNA 3

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X = me6-TG or O6-meG Reiterative processing 3 X T Recognition 5 and processing T Excision and resynthesis Aberrant recombinogenic DNA structure ATRATRIP CHEK1 G2 arrest 5 X 5 3 X 5

Misincorporation

EXO1 PolPCNA

Figure 3 | Dna mismatch repair and its contribution to potentially lethal Dna damage. a | Correction of Nature Reviews | Cancer replication errors. Erroneous incorporation of a non-complementary base by the replication apparatus (DNA polymerase plus the polymerase-associated proliferating cell nuclear antigen (PCNA) sliding clamp triggers recruitment of the mismatch recognition factor MutS (an MSH2 and MSH6 dimer). A second dedicated MMR factor MutL (a dimer of MLH1 and PMS2) is then recruited. The endonuclease activity of MutL introduces nicks that promote the loading of EXO1 on the 5 side of the misincorporated nucleotide. The 5-to-3 exonucleolytic activity of EXO1 excises a stretch of daughter strand of DNA including the mismatch. The extensive gap left by EXO1 is filled by DNA polymerase PCNA, which now incorporates the correct complementary nucleotide, and mismatch repair is completed by DNA ligase sealing the remaining nick. The key to successful correction is that the incorrect nucleotide resides, by definition, in the newly synthesized daughter DNA strand. b | The presence of a miscoding base analogue (X, for example, methyl 6-thioguanine (me6-TG) or O6-meG) in the template strand can confuse the MMR system. Here me6-TG derived by chemical S-methylation of 6-TG incorporated in an earlier round of replication, or O6-meG produced by a methylating agent, directs the incorporation of T. Me6-TG:T or O6-meG:T base pairs do not escape surveillance by MutSMutL, which triggers mismatch correction attempts that remain incomplete because of the impossibility of incorporating a perfectly paired base opposite the lesion28, 31. The anomalous DNA structures generated by the incomplete repair attempts can be observed as DNA strand interruptions34. During the S phase of the next cell cycle, these are converted into potentially lethal aberrant DNA structures that are substrates for recombination. These trigger activation of ATR (ataxia telangiectasia and Rad3-related)ATRIP (ATR-interacting protein) DNA-damage signalling, phosphorylation of CHEK1 and G2 cell-cycle arrest.

Similarly to 6-TG, DNA me6-TG codes ambiguously during replication and directs C and T insertion approximately equally44. DNA me6-TG:T base pairs are recognized particularly well, and significantly better than 6-TG-containing pairs, by MutS. This is fully consistent with their processing by MMR into potent, potentially lethal DNA lesions. one important implication of these findings is that the resistance of MMR-deficient cells provides them with a significant proliferative advantage over otherwise identical MMR proficient cells when challenged by a thiopurine or methylating agent. In laboratory studies,
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chronic exposure to escalating concentrations of either type of drug permits the selection of rare MMR-defective variants from a population of MMR-proficient cells29,48,49. This property may be important in the development of cancer in patients receiving thiopurines (see below). The enhanced resistance to haematological toxicity and death in MMR-deficient Msh2/ knockout mice treated with 6-MP is eloquent testimony to the direct involvement of MMR in the biological effects of thiopurines. Although inactivation of MMR is undoubtedly among the most important mechanisms by which cells acquire thiopurine resistance, it is by no means the only
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one. Alterations in TPMT or HPRT activity thiopurine transport into cells50 are also potential contributors. Whether the recently described interaction between DNA containing 6-TG:T (ReF. 51) or 6-TG:C (ReF. 52) and an apparent multiprotein complex containing glyceraldehyde-3-phosphate dehydrogenase has any significant role in determining the toxicity of thiopurines is currently uncertain. Purine biosynthesis. There is some evidence to suggest that the ability of 6-MP metabolites to inhibit de novo purine biosynthesis may contribute to their cytotoxic effects. In addition to the free thiopurine bases, TPMT also methylates both TIMP and TGMP18 (FIG. 2). The product of the former reaction, methylthioinosine monophosphate (meTIMP) is an efficient non-competitive inhibitor of phosphoribosylpyrophosphate amidotransferase (PPAT)53, which catalyses an early step in the pathway of purine biosynthesis. Inhibition reduces supplies of purine triphosphates for DNA and RNA synthesis, cellular metabolism and signalling, and is thought to compromise the clonal expansion of T cells. Despite early suggestions to the contrary24, the cytotoxic properties of thiopurines are still often thought to be a consequence of the depletion of purine nucleotides and their immunosuppressive effects are thought to reflect a particular reliance of T cells on the de novo purine synthesis rather than salvage. It should be noted, however, that effective salvage is an essential first step in thiopurine metabolism that precedes the formation of meTIMP. It is far from clear that PPAT inhibition is a major contributor to thiopurine-induced cytotoxicity and immunosuppression. The extreme myelotoxicity of thiopurines in patients provides the most direct evidence that meTIMP formation does not account for their cytotoxic properties. Myelotoxicity is associated with low, rather than high, TPMT and meTIMP levels54,55. Increased accumulation of 6-TG in DNA and RNA as a result of high TGN levels due to inadequate thiopurine catabolism provides a more likely explanation for toxicity. Indeed, the relationship between TGN levels and myelosuppression has been stressed many times56,57. If the immunosuppressive effects of thiopurines really are a consequence of reduced de novo purine synthesis, it follows that thiopurines must exert their cytotoxic and immunosuppressive effects through different mechanisms. Is it in fact necessary to postulate two mechanisms or might immunosuppression also be due to accumulation of TGNs and, ultimately, accumulation of 6-TG in DNA? Powerful inhibition of de novo purine synthesis is certainly immunosuppressive. This is exemplified by mycophenolate mofetil, a more recently developed immunosuppressant that is increasingly replacing azathioprine (FIG. 1). Mycophenolate mofetil is metabolized to mycophenolic acid (MPA) by plasma esterases. MPA is a powerful and selective inhibitor of inosine monophosphate dehydrogenase (IMPDH), which catalyses the rate-limiting step of GTP synthesis, and depletes the levels of GTP and dGTP58. It does not follow from this, however, that meTIMP inhibition of the non-rate-limiting PPAT in
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the same pathway underlies immunosuppression by 6-MP and azathioprine. As thiopurines have been extremely successful in the treatment of childhood leukaemia, many of the in vitro studies which implicated IMPDH inhibition by 6-MP metabolites were carried out with established tumour cell lines of T-cell origin. Unfortunately, many of the cell lines used in those studies have since been shown to be defective in DNA mismatch repair. These include the jurkat (MutS homologue 2 (MSH2)-deficient)59, CCFCeM (MutL homologue 1 (MLH1)-deficient)60, 293T (MLH1-deficient)61 and MoLT4 (PMS2-deficient)62 cell lines, all of which are atypically thiopurine-resistant because they lack the most significant executioner an active MMR system63. Thus, although overexpression of a transfected TPMT in CCF-CeM cells induced >99% inhibition of de novo purine synthesis, the changes in 6-TG and 6-MP sensitivity were small64. This suggests that the most significant mechanism of cytotoxicity is common to both thiopurines, involves TGN formation and 6-TG incorporation into DNA, and requires active mismatch repair. Clinical findings do not support a predominant role for de novo purine synthesis inhibition in cytotoxicity. Dervieux et al.65 showed that the rate of de novo purine synthesis in the T leukaemic cells of patients was unchanged by successful 6-MP treatment. By contrast, methotrexate, an acknowledged and potent inhibitor of nucleotide biosynthesis, was an extremely effective inhibitor and significantly enhanced the therapeutic effect of 6-MP. This suggests that 6-MP cytotoxicity towards leukaemic T cells is largely independent of any effect on de novo purine synthesis. Findings from mouse models support this conclusion. 6-MP treatment causes significantly less myelotoxicity in MMR-defective Msh2/ knockout mice than in their repair proficient Msh2+/+ or Msh2+/ counterparts. The phenotype of the TPMT knockout mouse66 provides more evidence that 6-MP and 6-TG share a common mechanism of toxicity. Despite producing no measurable meTIMP, Tpmt/ animals are much more sensitive to 6-MP than homozygous wild-type or heterozygous mice, in which meTIMP levels are more than 1,000fold higher. There seems little doubt that the toxic and myelosuppressive properties of 6-MP and 6-TG reflect the formation of TGNs and 6-TG incorporation into nucleic acids. Although the immune status of the animals was not addressed directly, there seems little reason to invoke an alternative to TGNs to explain the immunosuppressive properties of the thiopurines.

DNA 6-TG and UVA radiation The methylation in situ of DNA 6-TG described above is one example of its enhanced chemical reactivity. Its interaction with ultraviolet (UV) light provides a second example. The canonical DNA and RNA bases all absorb maximally in the UVC spectral region (~260 nm) and terrestrial life based on the conventional nucleic acids is possible only because removal of UVC from incident sunlight considerably reduces the threat of destructive photochemical damage to DNA. Thiopurines, by contrast,
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0.14 0.12 0.10 Absorbance 0.08 0.06 0.04
HN S N N N H

G 6-TG

0.02 0.00 0.00

H2N

6-TG

250

300 Wavelength (nm)

350

400

UVC (100280)

UVB

UVA (320400)

Figure 4 | absorbance spectra of 6-TG and G. Canonical DNA purines, for example, guanine (shown here), have no significant absorption of ultraviolet radiation at wavelengths longer than 300 nm. Replacement of the 6-O atom by sulphur Cancer see Nature Reviews | (6-TG chemical structure) causes the absorbance maximum to shift into the UVA region (~340 nm).

Chromophore
That part of a substance that absorbs visible light or, by extension, ultraviolet radiation.

Reactive oxygen species

(ROS). Highly unstable oxygencontaining chemical entities. ROS include oxygen free radicals such as the hydroxyl, peroxyl and superoxide anion radicals and non-free radical forms such as singlet oxygen. Low-level ROS production is an essential component of intracellular signalling.

Fenton-like reactions
Many of the most harmful changes in DNA are caused by hydroxyl radicals. In the Fenton reaction, the relatively innocuous hydrogen peroxide is converted into hydroxyl radicals in a reaction involving iron associated with DNA.

Tm

The temperature at which a DNA double helix dissociates into single strands.

are significant UVA chromophores and both 6-TG and 6-MP have an absorbance maximum at approximately 340 nm (FIG. 4). As UVA comprises over 90% of solar ultraviolet radiation that reaches earth, 6-TG in DNA is a potential hazard. Because of its longer wavelengths, UVA radiation is not effectively eliminated by glass and it also penetrates significantly deeper into skin. As much as 2030% of UVA incident at the skin surface penetrates to the lower layers that contain the stem cells responsible for skin renewal67. For a general review of the health aspects of ultraviolet radiation, see ReF. 68. In aqueous solution, both 6-TG69 and 6-MP70 are Type I and Type II UVA photosensitizers that generate highly damaging reactive oxygen species (RoS) when irradiated in the presence of molecular oxygen. In a Type I reaction, the thiopurine absorbs UVA energy and is converted to an unstable excited triplet state that can interact with oxygen to form a thiopurine radical and superoxide (o2). Fenton-like reactions convert o2 into the hydroxyl radicals (oH) that are a source of damage to cellular macromolecules. Type II reactions involve transfer of the absorbed radiation energy directly to ground-state triplet molecular oxygen (3o2) to generate singlet oxygen (1o2) a non-radical with a longer half-life than most RoS. Both oH and 1o2 cause potentially mutagenic damage to DNA. This includes breakage of the DNA strands as well as oxidation-induced changes to constituent bases and deoxyribose. 1o2 is generally considered to be the predominant damaging species produced in cells exposed to relatively high fluences of UVA71. In this case, the photochemical generation of 1o2 is likely to involve endogenous cellular UVA chromophores that have yet to be fully characterized. Among RoS, 1o2 is particularly

implicated in protein damage in the form of crosslinks between amino acids72,73. Its significantly longer half-life (~110 s in aqueous environments) gives it a larger sphere of action than the more unstable RoS. As well as being a source of reactive oxygen, 6-TG itself is also highly susceptible to oxidation by RoS. In particular, low doses of UVA cause a rapid loss of its characteristic absorbance at 340 nm. Two major photoproducts have been identified following UVA irradiation of 6-TG in solution69,22. The first of these, guanine-6-thioguanine (2-amino-6-(2-amino-6-purinyl)-thiopurine, GSG) is a 6-TG dimer that is formed in a bimolecular reaction and is unlikely to be a significant DNA photoproduct. oxidation of the sulphur atom occurs to generate the second major product, the highly fluorescent guanine6-sulphonate (GSo3, FIG. 5). GSo3 is formed to some extent by Type I photosensitization and its formation is partially inhibited by free radical scavengers. Type II reactions involving 1o2 are probably the more significant contributor, however, and visible light in the presence of Rose Bengal a source of 1o2 efficiently converts 6-TG to GSo3. Analogous reactions occur in DNA, and DNA GSo3 is formed when either single-stranded or double-stranded DNA containing 6-TG is irradiated with UVA or treated with a mild chemical oxidizing agent74. Cells containing DNA substituted with a low level of 6-TG are susceptible to the same photochemical reactions. Low UVA doses are sufficient to produce a burst of RoS within cellular 6-TG-substituted DNA and to convert a considerable fraction of the 6-TG to GSo3 (ReF. 69). Generation of RoS inside the DNA molecule itself is potentially catastrophic. In addition to oxidizing 6-TG to GSo3, RoS can inflict collateral damage on surrounding normal DNA bases75, cause sugar modifications, introduce DNA strand breaks and damage DNA-associated proteins (FIG. 5). All of these are potentially detrimental to cellular wellbeing. It is likely that RoS damage to cellular DNA is reflected in the synergistic cytotoxicity and mutagenicity of 6-TG and UVA69. GSo3 is a negatively charged and bulky DNA adduct. It has a considerable influence on DNA stability and profoundly reduces the Tm of duplex oligodeoxynucleotides22. Helix destabilization is largely independent of the base in the opposing strand, and DNA GSo3 is incapable of forming stable pairs with any of the canonical DNA bases. It is therefore unsurprising that a template GSo3 is a severe block to primer extension by DNA polymerases and can be bypassed only by Y-family DNA polymerases, which have a relatively relaxed stringency76. DNA 6-TG and UVA combine with analogous effect in living cells. Low doses of UVA cause rapid inhibition of DNA replication in cells with DNA 6-TG. This triggers monoubiquitylation of the proliferating cell nuclear antigen (PCNA) replication factor generally regarded as a signal for the recruitment of specialized DNA polymerases to bypass replication-blocking DNA lesions. The same treatments also induce the p53-dependent DNA damage response22. Stabilization of the p53 protein and induction of p21 (also known as CDKN1A) occur with the expected kinetics, but at higher UVA doses the transcriptional response of
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ROS Radical OH hydroxyl O superoxide 2 O R peroxyl 2 OR alkoxyl NO nitric oxide Non-radical 1O singlet oxygen 2 ONOO peroxynitrite H2O2 hydrogen peroxide
O O N H 2N N S O N N dR HN O N dR

Possible detrimental effects of intracellular ROS Lipid peroxidation Protein damage (1O2) Aldehydes Amino-acid crosslink Double-strand/ single-strand breaks DNA adducts Damage to DNAassociated proteins

DNA damage

Oxidized bases
O

CH3 OH OH HN H2N

H N O N dR

dGSO3

Thymidine glycol

Oxo8deoxyguanosine

Figure 5 | reactive oxygen species (rOS) and potential damage to cellular macromolecules. Radical and nonNature Reviews | Cancer radical forms of ROS can cause direct damage to DNA constituents in the form of oxidized bases and deoxyribose, and cleavage of the phosphodiester backbone to form single- or double-strand breaks. They can also initiate reactions that generate secondary reactive species, such as aldehydes, that can also form DNA lesions. DNA 6-thioguanine is a particular target for oxidation and one of the major products (GSO3) is shown. Proteins are also vulnerable to ROS, particularly to 1O2 which reacts with aromatic, basic and sulphur-containing amino acids.

Y-family DNA polymerases

Y-family DNA polymerases have a more open active site than A- and B-family DNA polymerases and can accommodate covalently modified template DNA bases. This allows them to insert nucleotides opposite damaged bases. This bypass mode allows the replication of damaged DNA to continue, although often at a cost to fidelity.

p21 is attenuated, suggesting direct inhibitory effects of DNA damage on transcription. The combined effects of 6-TG and UVA on replication and transcription are consistent with the introduction of bulky DNA adducts and probably reflect the formation of DNA GSo3. DNA is not the only target of photochemically generated RoS. Photochemical protein damage principally by 1o2, which is formed in significant amounts during the photochemical destruction of 6-TG is a contributor to several pathological conditions including lens degeneration in the eye and altered dermal collagen function77,78. Crosslinked or aggregated proteins are major products of intracellular 1o2, which can react with aromatic (phenylalanine, tyrosine and tryptophan), basic (arginine, lysine and histidine) and sulphur-containing (methionine and cysteine) amino acids. Among these, histidine is the major casualty with the highest rate constant for reaction with 1 o2 and histidine appears to be essential for 1o2-mediated protein crosslinking79. The generation of 1o2 from DNA 6-TG and UVA creates what might be a unique danger for proteins intimately associated with DNA. Although 1o2 molecules have a relatively long half-life (of the order of microseconds rather than the nanoseconds of other RoS), they react close to their site of formation. The multiprotein replication and transcription complexes as well as histones and non-histone DNA binding proteins are all potential targets for oxidation. PCNA is one casualty of DNA-generated 1o2. This important DNA replication and repair factor is a homotrimeric complex that acts as a sliding clamp to tether DNA polymerases to DNA, thereby improving their processivity and accuracy. After UVA treatment of cells containing DNA 6-TG, the subunits of PCNA engaged in replicating thiopurinesubstituted DNA become covalently bound to one another74. The precise structure of the PCNA complex

and the biological consequences of its formation have yet to be determined. Covalent linkage between the subunits of the PCNA trimer is likely to compromise its function, however, because loading of PCNA onto DNA requires transient opening of the trimer. In addition, there is no reason to suppose that PCNA is the only oxidized protein and its covalent modification illustrates the vulnerability of DNA-associated proteins in cells that are exposed to 6-TG and UVA. RoS-mediated DNA damage is a major source of mutation. Cells are adapted to deal with the threat from the RoS that are inevitably generated during normal aerobic metabolism. Conditions that significantly exceed these levels saturate cellular defences and induce oxidative stress. The photochemical reactions of DNA 6-TG are potentially hazardous for two reasons. First, although high levels of UVA alone generate RoS, DNA 6-TG is a significant UVA sensitizer and may reduce the UVA dose that is required to cause damage to within the range of normal sunlight exposure. Second, the RoS are generated in DNA itself. As these are likely to react close to their site of formation, DNA, and associated proteins responsible for its accurate replication, are front-line targets. A plausible outcome of the photochemical reactions of DNA 6-TG is an increased burden of mutation and an increased risk of malignancy.

Clinical implications therapy-related cancer Azathioprine is designated as a human carcinogen largely on the basis of epidemiological evidence. Although unequivocal, the data provide no indication as to the mechanism by which cancers might arise. In the following sections we consider how the properties of azathioprine, in particular of DNA 6-TG, might influence the development of particular malignancies.
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Acute myeloid leukaemia. Myelosuppression is doselimiting for thiopurine treatment and doses are adjusted to the maximal level compatible with maintaining satisfactory blood cell counts. Thiopurine treatment places the particularly sensitive bone marrow cells under a selective pressure that mimics laboratory experiments designed to select rare MMR-defective cells29,48,49. Therapy-related AML is a potential complication of thiopurine therapy for acute lymphoblastic leukaemia80,81 and is also overrepresented among organ transplant patients. The relative risk is 5.5 for heart and lung transplants and 2.1 for cadaver kidney recipients49. Unlike the cancers that are known to involve viruses82, AML is late in onset and the incidence only diverges significantly from that of a matched immunocompetent population after a lag of ~35 years49. The azathioprine-dose dependency in both transplant groups suggests a causal contribution from the thiopurine. It is particularly noteworthy that the frequency of microsatellite instability (MSI) is high among transplant-related AML cases. MSI is generally rather infrequent (<5% of cases) in de novo AML unrelated to therapy. Seven out of seven transplant-related cases were MSI+, however, suggesting that the frequency of MSI among transplantrelated AML is likely to be closer to the ~50% seen in AML after chemotherapy49. A possible explanation for the high frequency of mismatch repair defects in immunosuppression-related AML is the selective outgrowth of myeloid precursor cells with thiopurine-resistance acquired by somatic inactivation of the MMR pathway. Cells with MMR defects have highly increased rates of spontaneous mutation because replication errors remain uncorrected. This mutator phenotype would accelerate the development of malignancy (see FIG. 3). Skin cancer. Skin cancer, the most significantly overrepresented malignancy in transplant patients, develops after ~10 years of immunosuppression83,84, although this is dependent on factors such as age at transplant. The relative risk for SCC among transplant patients is up to 250, with a more modest, although still considerable, tenfold increased risk of basal cell carcinoma85,86. Several factors probably contribute to this striking increase in SCC. It is plausible that the human papillomaviruses some of which have oncogenic potential may be involved. However, their role, if any, in transplant-related skin cancer remains undefined5. epidemiological evidence comprising geographical incidence, lifestyle factors and tumour distribution on exposed regions of the body (face, scalp, forearms and backs of hands) identifies sunlight exposure as an important causative factor87,88. It is possible to imagine sunlight contributing to increased risk in two ways, which are not mutually exclusive. Ultraviolet radiation is immunosuppressive and might simply increase risk by locally intensifying immunosuppression89,90. As an alternative we suggest that the photochemical reaction of DNA 6-TG with UVA in keratinocytes which generates RoS is a potentially mutagenic process that might ultimately contribute to SCC pathogenesis. In transplant patients, long-term systemic immunosuppression with azathioprine results in detectable steady-state DNA 6-TG in skin cells69 as well as lymphocytes91,92. Although the extent of substitution would not be expected to generate sufficient DNA me6-TG to trigger MMR-related toxicity, the levels of DNA 6-TG would still be expected to confer UVA sensitivity. Indeed, the skin of patients taking azathioprine is hypersensitive to ultraviolet light and their minimal erythema dose (MeD) the dose needed to produce mild sunburn on skin that is not normally exposed to sunlight is significantly reduced for UVA69. Their MeD for UVB is unchanged. erythema is linked to DNA damage and the cutaneous UVA hypersensitivity of these patients is consistent with the introduction of replication and transcription blocking 6-TG DNA photoproducts. 6-TG and UVA treatment is mutagenic in cultured cells in the laboratory. It is possible that DNA damage produced by the RoS generated photochemically from DNA 6-TG could be a significant factor in the extremely high incidence of skin cancer in immunosuppressed organ transplant patients. In view of this, transplant patients are usually looked after in dedicated dermatology clinics, and patients treated with thiopurines should be educated in the need for sensible sun protection, should report any skin lesions that develop and should be examined at appropriate intervals. Chronic inflammatory disorders. The particular susceptibility of DNA 6-TG to chemical oxidation suggests that the growing number of patients who receive azathioprine for inflammatory disorders may have an increased risk of developing cancer. We will illustrate this with the example of inflammatory bowel disease (IBD) but, in principle, the considerations apply to any chronic inflammatory condition. IBD, comprising the two major sub-phenotypes ulcerative colitis and Crohns disease, is a chronic relapsing inflammatory disorder. The pathological hallmark is a dysregulated intestinal immune response leading to the development of unmitigated mucosal inflammation. It is generally accepted that these disorders reflect the exaggerated response of a genetically predisposed individual to microbial or host antigens93. In some patients, particularly with recalcitrant or corticosteroid-dependent IBD, immunosuppression with azathioprine or 6-MP is clinically effective. There is some concern that this thiopurine treatment might increase the risk of cancer, primarily colorectal carcinoma and lymphoma, but published data are conflicting94,95. The discrepancies might reflect different ranges of treatment duration, patient age and disease severity, and the risks of long-term treatment might have been underestimated. In view of the susceptibility of DNA 6-TG to oxidation, it is reasonable to suspect that azathioprine or 6-MP treatment might confer additional cancer risk in IBD patients. The relationship between colorectal carcinoma and IBD is unequivocal and lifetime risk correlates proportionately with the length, severity and extensiveness of the inflammation of the gastrointestinal mucosa. Although the exact mechanism remains incompletely defined, sustained oxidative stress an excess of RoS consequent to pathological inflammation almost certainly contributes to carcinogenesis 96. This conclusion is supported by data from knockout mice in which simultaneous ablation of glutathione peroxidases 1 and 2 (RoS-quenching enzymes)
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Ulcerative colitis
A form of inflammatory bowel disease in which inflammation affects the large intestine or colon and consists of characteristic ulcers or open sores.

Crohns disease
A type of inflammatory bowel disease. It is characterized by inflammation across the entire wall of the affected mucosa and can affect any part of the gastrointestinal tract.

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results in ileocolic inflammation, increased tumours and increased frequencies of oxidative stress-related signature mutations97. The gastrointestinal mucosa in IBD is characterized by the presence of activated immune cells. Phagocytic cells, principally neutrophils and macrophages, produce RoS and other metabolites that cause pathological tissue destruction. Proliferating colonic epithelial cells within this intense inflammatory microenvironment are likely to be susceptible to the mutagenic effects of chronically generated RoS. In order to generate potentially harmful DNA oxidation products phagocyte-derived RoS must be able to diffuse across cellular membranes. H2o2 seems the most likely effector molecule as it can generate DNA damaging oH radicals by the Fenton reaction. DNA damage by the products of lipid peroxidation is also possible. In view of the particular susceptibility of DNA 6-TG to oxidation and the demonstrated mutagenicity of combined DNA 6-TG and RoS, it seems possible that the milieu of oxidative stress may represent a particular mutational hazard for DNA 6-TG-containing colonic epithelial cells of patients treated with azathioprine. Although these concerns still require validation by detailed clinical studies, it seems reasonable to ensure that patients treated with thiopurines for IBD receive long-term surveillance for possible colorectal cancer. of use is a testimony to their success. Following a more detailed understanding of thiopurine metabolism and the development of pharmacogenetic testing it is anticipated that their safety profile, particularly with respect to myelotoxicity, will continue to improve. It is undisputed that thiopurine treatment is associated with cancer. This is clearly established for haematological and virus-associated malignancies, and skin cancers. We have described how the presence of the thiobase alters the chemistry of DNA and how this might facilitate the development of cancer. one postulated route involves the increased rate of spontaneous mutation that accompanies inactivation of the MMR system. An alternative route invokes oxidation and oxidative stress, a widespread condition that is particularly prevalent in inflammatory disorders. We would emphasize, however, that the individual patient risk is likely to be small, and in many cases is far outweighed by the benefits of treatment. This is obvious in the case of life-saving organ transplantation. In the case of chronic non-life-threatening conditions such as rheumatoid arthritis, the equation is perhaps less clear-cut. In any event, the potential complication of therapy-related cancer should not be ignored. The past few years have seen an explosion in the development of novel immunosuppressants, many of which are replacing some of the more established treatments such as azathioprine and ciclosporin (FIG. 1). The modes of action of these newer drugs, insofar as they are known in detail, differ significantly from that of the thiopurines. They have not been in clinical use for long and the implications for cancer development cannot be known at this early stage. In view of the long-term nature of immunosuppression, continued awareness of therapy-related cancer as a possible delayed side effect of treatment seems prudent.
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Concluding remarks over the past five decades countless patients, many suffering from chronic debilitating diseases, have benefited from treatment with thiopurines, and the use of azathioprine as an immunosuppressant was instrumental in making organ transplantation from unrelated donors a reality. In the modern era of pharmacotherapy this longevity
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We are indebted to the numerous laboratory colleagues, past and present, who helped shape many of these opinions, especially to J. Offman, P. ODonovan and C. Perrett, who contributed particularly to our studies of therapy-related cancer. Many of the ideas expressed in this article have been developed and refined over several years. We thank M. Bignami, Y.-Z. Xu, P. Swann, G. Opelz, C. Harwood and J. McGregor for their significant contributions to this.

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DATABASES
Entrez Gene: http://www.ncbi.nlm.nih.gov/entrez/query. fcgi?db=gene ATR | ATRIP | CDKN1A | CHEK1 | HPRT1 | IMPDH | MLH1 | MSH2 | MSH6 | p53 | PCNA | PMS2 | PPAT |TPMT

FURTHER INFORMATION
Peter Karrans homepage: http://science.cancerresearchuk. org/research/loc/london/lifch/karranp/
all lInkS are acTIve In The OnlIne pDf

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www.nature.com/reviews/cancer