Reversal of UVA skin photosensitivity and DNA damage in kidney transplant recipients by replacing azathioprine.

Azathioprine is associated with enhanced skin photosensitivity to ultraviolet A (UVA) and leads to incorporation of 6-thioguanine (6-TG) into DNA of dividing cells. Unlike canonical DNA, 6-TG DNA is damaged by UVA, which comprises more than 90% of the ultraviolet reaching earth. Skin photosensitivity to UVA and UVB was measured in 48 kidney transplant patients immunosuppressed either by azathioprine (n = 32) or mycophenolate (n = 16). In 23 patients, azathioprine was subsequently replaced by mycophenolate and skin photosensitivity, DNA 6-TG content in peripheral blood mononuclear cells, and susceptibility to UVA-induced DNA damage were monitored for up to 2 years. The mean minimal erythema dose to UVA on azathioprine was twofold lower than on mycophenolate. Three months after replacing azathioprine by mycophenolate mofetil, the minimal erythema dose to UVA had increased from 15 to 25 J/cm(2) (p < 0.001) accompanied by reduced DNA 6-TG content. P53 protein expression in irradiated skin indicated reduced susceptibility to UVA-induced DNA damage. 6-TG DNA in peripheral blood mononuclear cells remained measurable for over 2 years. Replacing azathioprine selectively reduced the skin photosensitivity to UVA, attenuated UVA-induced skin DNA damage, and is likely based on incorporated 6-TG in DNA.

Thiothymidine combined with UVA as a potential novel therapy for bladder cancer.

BACKGROUND: Thiothymidine (S(4)TdR) can be incorporated into DNA and sensitise cells to DNA damage and cell death following exposure to UVA light. Studies were performed to determine if the combination of S(4)TdR and UVA could be an effective treatment for bladder cancer. METHODS: Uptake and incorporation of S(4)TdR was determined in rat and human bladder tumour cell lines. Measures of DNA crosslinking and apoptosis were also performed. In vivo activity of the combination of S(4)TdR and UVA was investigated in an orthotopic model of bladder cancer in rats. RESULTS: Thiothymidine (200 µM) replaced up to 0.63% of thymidine in rat and tumour bladder cancer cells. The combination of S(4)TdR (10-200 µM) and UVA (1-5 kJ m(-2)) caused apoptosis and cell death at doses that were not toxic alone. Addition of raltitrexed (Astra Zeneca, Alderley Edge, Cheshire, UK) increased the incorporation of S(4)TdR into DNA (up to 20-fold at IC(5)) and further sensitised cells to UVA. Cytotoxic effect was associated with crosslinking of DNA, at least partially to protein. Intravenous administration of S(4)TdR, in combination with UVA delivered directly to the bladder, resulted in an antitumour effect in three of five animals treated. CONCLUSION: These data indicate that the combination of S(4)TdR and UVA has potential as a treatment for bladder cancer, and give some insight into the mechanism of action. Further work is necessary to optimise the delivery of the two components.

Expression of DNA mismatch repair proteins and MSH2 polymorphisms in nonmelanoma skin cancers of organ transplant recipients.

BACKGROUND: Organ transplant recipients (OTRs) have an increased risk of skin cancer. Treatment with azathioprine, commonly used in post-transplant immunosuppressive regimens, results in incorporation of 6-thioguanine (6-TG) into DNA. Mismatch repair (MMR)-defective cells are resistant to killing by 6-TG. Azathioprine exposure confers a survival advantage on MMR-defective cells, which are hypermutable and may therefore contribute to azathioprine-related nonmelanoma skin cancer, a phenomenon we have previously demonstrated in transplant-associated sebaceous carcinomas. The MSH2 protein is an important component of DNA MMR. The -6 exon 13 T>C MSH2 polymorphism is associated with impaired MMR, drug resistance and certain cancers. OBJECTIVES: To investigate (i) whether loss of MMR protein expression and microsatellite instability are over-represented in squamous cell carcinomas (SCCs) from OTRs on azathioprine compared with SCCs from immunocompetent patients, and (ii) whether the MSH2 -6 exon 13 polymorphism is over-represented in OTRs with skin cancer on azathioprine. METHODS: (i) Immunohistochemical staining was used to assess expression of the MMR proteins MSH2 and MLH1 in cutaneous SCCs from OTRs on azathioprine and from immunocompetent patients. (ii) Blood samples from OTRs on azathioprine with and without skin cancer were genotyped for the -6 exon 13 MSH2 polymorphism. RESULTS: (i) MSH2 and MLH1 protein expression was not altered in SCCs from OTRs on azathioprine and there was no difference in expression between SCCs from OTRs and immunocompetent patients. (ii) There was no association between MSH2 polymorphism genotype frequency and OTR skin cancer status. CONCLUSIONS: Despite previous findings in transplant-associated sebaceous carcinomas, defective MMR and the -6 exon 13 MSH2 polymorphism are unlikely to play a significant role in the development of SCC in OTRs on azathioprine.

Developmental decline in DNA repair in neural retina cells of chick embryos. Persistent deficiency of repair competence in a cell line derived from late embryos.

Neural retinas of 6-day-old chick embryos synthesize DNA and are able to carry out DNA excision repair. However, in contrast to the situation in human cells, the maximum rate of repair induced by N-acetoxy acetylaminofluorene (AAAF) is no greater than that induced by methyl methanesulfonate (MMS). With advancing differentiation of the retina in the embryo, cell multiplication and DNA synthesis decline and cease, and concurrently the cells lose the ability to carry out DNA excision repair. Thus, in 15-16-day embryos, in which the level of DNA synthesis is very low, DNA repair is barely detectable. If retinas from 14-day embryos are dissociated with trypsin and the cell suspension is plated in growth- promoting medium, DNA synthesis is reinitiated; however, in these cultures there is no detectable repair of MMS-induced damage, and only low levels of repair are observed after treatment with AAAF. A cell line was produced, by repeated passaging of these cultures, in which the cell population reached a steady state of DNA replication. However, the cell population remained deficient in the ability to repair MMS-induced damage. This cell line most likely predominantly comprises cells of retino-glial origin. Possible correlations between deficiency in DNA repair mechanisms in replicating cells and carcinogenesis in neural tissues are discussed.

PTCH mutations in basal cell carcinomas from azathioprine-treated organ transplant recipients.

The immunosuppressant azathioprine is used to prevent graft rejection after organ transplantation. To investigate whether azathioprine-associated mutagenesis contributes to the high incidence of skin tumours in organ transplant recipients (OTRs), we analysed PTCH gene mutations in 60 basal cell carcinomas (BCC); 39 from OTRs receiving azathioprine and 21 from individuals never exposed to azathioprine. PTCH was mutated in 55% of all tumours, independent of azathioprine treatment. In both the azathioprine and non-azathioprine groups, transitions at dipyrimidine sequences, considered to indicate mutation by ultraviolet-B radiation, occurred frequently in tumours from chronically sun-exposed skin. In BCC from non-sun-exposed skin of azathioprine-treated patients, there was an over-representation of unusual G:C to A:T transitions at non-dipyrimidine sites. These were exclusive to the azathioprine-exposed group and all in the same TGTC sequence context at different positions within PTCH. Meta-analysis of 247 BCCs from published studies indicated that these mutations are rare in sporadic BCC and had never previously been reported in this specific sequence context. This study of post-transplant BCC provides the first indication that azathioprine exposure may be associated with PTCH mutations, particularly in tumours from non-sun-exposed skin.

DNA double strand break repair in mammalian cells.

Human cells can process DNA double-strand breaks (DSBs) by either homology directed or non-homologous repair pathways. Defects in components of DSB repair pathways are associated with a predisposition to cancer. The products of the BRCA1 and BRCA2 genes, which normally confer protection against breast cancer, are involved in homology-directed DSB repair. Defects in another homology-directed pathway, single-strand annealing, are associated with genome instability and cancer predisposition in the Nijmegen breakage syndrome and a radiation-sensitive ataxia-telangiectasia-like syndrome. Many DSB repair proteins also participate in the signaling pathways which underlie the cell's response to DSBs.

DNA excision repair pathways.

The major DNA excision repair pathways of base excision repair for endogenous DNA lesions and nucleotide excision repair for DNA damage inflicted by ultraviolet light have been reconstructed with purified mammalian proteins and details of these repair mechanisms are emerging. Similar data are becoming available with regard to mismatch repair for correction of replication errors. Deletion of individual DNA repair proteins in knockout mice provides information on the roles of such factors in vivo and recent three-dimensional structures of several repair enzymes explain their detailed modes of action.

Azathioprine treatment photosensitizes human skin to ultraviolet A radiation.

BACKGROUND: Azathioprine is used to treat a variety of conditions and to prevent graft rejection in organ transplant recipients (OTRs). OBJECTIVES: To investigate clinically our previous finding that azathioprine metabolites interact with ultraviolet (UV) A radiation to form promutagenic oxidative DNA damage and to determine whether this may be causal or contributory to the development of excess skin cancers post-transplantation. METHODS: The clinical corollary of these data were investigated. Five patients were recruited and the minimal erythema dose (MED) for UVB, UVA and solar-simulated radiation (SSR) was determined for each person before, and at least 12 weeks after, starting azathioprine therapy. RESULTS: In all five patients azathioprine treatment was associated with an increased UVA and SSR sensitivity of the skin and a significant reduction in MEDs for UVA and SSR. We found no change in UVB-induced erythema or MED. In addition, we found that DNA from the skin of patients on azathioprine contains 6-thioguanine (6-TG). CONCLUSIONS: Our findings confirm the presence of DNA 6-TG in the skin of those taking therapeutic doses of azathioprine and provide support for the hypothesis that DNA damage occurs when DNA 6-TG interacts with UVA, resulting in abnormal cutaneous photosensitivity.

Photoactivation of DNA thiobases as a potential novel therapeutic option.

The thiopurines, 6-thioguanine and 6-mercaptopurine, are antileukemic agents that are incorporated into DNA following retrieval by the purine salvage pathway (see [1] for a review). Their toxicity requires active DNA mismatch repair (MMR), and thiopurine resistance is an acknowledged phenotype of MMR-defective cells [2, 3]. In addition to these direct cytotoxic effects, DNA thiobases have distinctive photochemical properties [4], the therapeutic potential of which has not been extensively evaluated. We report here that the thiopyrimidine nucleoside 4-thiothymidine is incorporated into DNA. It does not induce MMR-related toxicity, but it interacts synergistically with UVA light and dramatically sensitizes cultured human cells to very low, nonlethal UVA doses. 4-thiothymidine induced UVA dose enhancements of around 100-fold in DNA repair-proficient cells. Nucleotide excision repair-defective xeroderma pigmentosum cells were sensitized up to 1000-fold, implicating bulky DNA photoproducts in the lethal effect. The synergistic action of thiothymidine plus UVA required thymidine kinase, indicating a selective toxicity toward rapidly proliferating cells. Cooperative UVA cytotoxicity is a general property of DNA thiobases, and 6-thioguanine and 4-thiodeoxyuridine were also UVA sensitizers. Thiobase/UVA treatment may offer a novel therapeutic approach for the clinical management of nonmalignant conditions like psoriasis or for superficial tumors that are accessible to phototherapy.

Processing of O6-methylguanine by mismatch correction in human cell extracts.

Human cell extracts perform an aberrant form of DNA synthesis on methylated plasmids [1], which represents processing of O6-methylguanine (O6-meG). Here, we show that extracts of colorectal carcinoma cells with defects in the mismatch repair proteins that normally correct replication errors do not carry out this synthesis. hMSH2-defective LoVo cell extracts (hMSH for human MutS homologue) performed O6-meG-dependent DNA synthesis only after the addition of the purified hMutS alpha mismatch recognition complex. Processing of O6-meG by mismatch correction requires PCNA and therefore probably DNA polymerase delta and/or epsilon. Mismatch repair-defective cells withstand O6-meG in their DNA [2], making them tolerant to methylating agents. Methylation-tolerant HeLaMR clones, with a mutator phenotype and a defect in either mismatch recognition or correction in vitro, also performed little O6-meG-dependent DNA synthesis. Assays of pairwise combinations of tolerant and colorectal carcinoma cell extracts identified hMLH1 as the missing mismatch repair function in a group of tolerant clones. The absence of processing by extracts of methylation-tolerant cells provides the first biochemical evidence that lethality of DNA O6-meG derives from its interaction with mismatch repair.

Selection for beta 2-microglobulin mutation in mismatch repair-defective colorectal carcinomas.

Novel peptide antigens complexed with human leukocyte antigen (HLA) and beta 2-microglobulin (beta 2M) molecules are presented at the cell surface to cytotoxic T lymphocytes (CTLs), provoking lysis of the antigen-presenting cell [1]. In tumor cells, genetically altered or abnormally expressed proteins provide a source of peptides that can be presented to CTLs; the resulting anti-tumour CTL responses may provide part of the body's defence against cancer. Disabling mutations in the HLA and beta 2M proteins required for peptide presentation allow a tumour cell to escape destruction by CTLs. Cells with deficient DNA mismatch repair have high spontaneous mutation rates [2] and produce many altered proteins that are a potential source of numerous unique peptides. Mutator tumour cells might therefore be particularly vulnerable to immune surveillance and CTL attack. Mutator phenotypes [3,4] and loss of beta 2M (or HLA) expression [5,6] are both relatively common among sporadic colorectal tumours. We have compared the frequency of beta 2M mutations in sporadic colorectal and other tumours with and without a mutator phenotype. Mutations were more frequent among colorectal tumours with the microsatellite instability indicative of a defect in DNA mismatch repair. The inactivating beta 2M mutations were predominantly frameshifts, which is consistent with the underlying mismatch repair defects. Evasion of immune surveillance by acquiring beta 2M mutations therefore occurs at high frequency in tumour cells with a mutator phenotype due to defective DNA mismatch repair.

DNA mismatch binding defects, DNA damage tolerance, and mutator phenotypes in human colorectal carcinoma cell lines.

DNA mismatch binding in vitro, resistance to DNA methylation damage, and spontaneous mutation rates were examined in human colorectal adenocarcinoma cell lines. Of 11 cell lines, 3 (DLD1, HCT15, and LoVo) were defective in mismatch binding. All three lines had a mutator phenotype. These properties indicate that DLD1 and HCT15 may, like LoVo, carry mutations in the mismatch recognition protein hMSH2. Mismatch binding was normal in the remaining eight lines, including HCT116 in which a second mismatch repair protein, hMLH1, is defective. Two lines, SW620 and SW48, did not express detectable levels of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. SW620 exhibited the expected sensitivity to N-methyl-N-nitrosourea. In contrast, SW48 cells were highly resistant to N-methyl-N-nitrosourea and also slightly to methyl methanesulfonate, indicating that they are tolerant to DNA methylation damage. SW48 exhibited the spontaneous mutator phenotype and microsatellite instability that are hallmarks of a defect in mismatch repair. This cell line provides evidence for the association between methylation tolerance and defective mismatch correction in human colorectal carcinoma cells. The properties of methylation-tolerant, mismatch repair-defective cells identify possible selective pressures that might facilitate the natural selection of mismatch repair-defective tumors.

Epigenetic silencing of the DNA repair enzyme O6-methylguanine-DNA methyltransferase in Mex- human cells.

Regulation of the expression of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) has been investigated in a number of human lymphoblastoid cell lines. In a number of Mex- cell lines that do not express methyltransferase activity, CpG sequences in the mgmt gene were hypomethylated with respect to methyltransferase-expressing Mex+ lines. In the cell line GM1953(S), in which the mgmt gene is coregulated with the thymidine kinase and galactokinase genes, reexpression of all three activities was experimentally induced. In this case, the mgmt gene in the nonexpressing cells was found to be hypermethylated and underwent a demethylation at CpG sequences that was coincident with the reappearance of the mgmt mRNA and the three enzyme activities. The simultaneous silencing of three activities in these cells was correlated with an increase in DNA 5-methylcytosine that was widespread throughout the genome. The data indicate that MGMT expression can be controlled epigenetically in human lymphoid cell lines, although the relationship between cytosine methylation and MGMT expression is complex. Furthermore, the rapid alterations in methylation in GM1953(S) cells indicate the existence of signals that can induce widespread and abrupt alterations in cytosine methylation in human cells in culture.

O6-Methylguanine-DNA methyltransferase of human lymphoid cells: structural and kinetic properties and absence in repair-deficient cells.

Human lymphoid cell lines contain a DNA repair enzyme which removes the mutagenic alkylation lesion O6-methylguanine from DNA. The enzyme transfers the methyl group to a protein cysteine residue, generating S-methylcysteine, and is inactivated as a consequence of the reaction. Apparently the methylated enzyme represents a dead-end complex. The transfer reaction is very rapid and is completed in less than 1 min at 37 degrees, but methyl group transfer from single-stranded DNA or heavily damaged DNA is less efficient. The active methyltransferase and the methylated protein both have molecular weights of 21,000 to 22,000, as determined by gel filtration. Lymphoid cell lines proficient in repair of O6-methylguanine in vivo, Mex+, contain 10,000 to 25,000 molecules of the methyltransferase per cell. In contrast, repair-deficient cell lines, Mex-, do not contain detectable amounts of the enzyme. The latter point was verified by applying a partial purification procedure for the enzyme to cell-free extracts from two Mex- cell lines.

DNA excision-repair deficiency of human peripheral blood lymphocytes treated with chemical carcinogens.

Human peripheral blood lymphocytes stimulated with concanavalin A for 72 hr have a 10-fold greater capacity to repair DNA damage induced by N-acetoxy-2-acetylaminofluorene than do unstimulated cells. The increased capacity of concanavalin A-activated cells to repair DNA is not observed after 24 hr in culture, a time at which stimulated cells have not begun to synthesize DNA. The maximum rate of repair synthesis obtained after treatment of stimulated cells with the "large patch"-inducing agent, N-acetoxy-2-acetylaminofluorene, is twice that obtained with methyl methanesulfonate, an agent inducing "small patch" repair. The difference between the maximum rates obtained with N-acetoxy-2-acetylaminofluorene and methyl methanesulfonate is 6-fold in a human lymphoblastoid line. Unstimulated lymphocytes show almost identical rates of repair after treatment with either N-acetoxy-2-acetylaminofluorene or methyl methanesulfonate. There is close correlation between the rate of N-acetoxy-2-acetylaminofluorene-induced repair synthesis and the loss of acetylaminofluorene adducts from DNA. Treatment of lymphocytes with methyl methanesulfonate leads to degradation of cellular DNA with the production of single-stranded regions. Such degradation is not observed with N-acetoxy-2-acetylaminofluroene. We conclude that the rate of excision repair is a function of the capacity of cells for DNA synthesis and that lymphocytes that do not synthesize DNA have a limited repair capacity and cannot be used to distinguish between large and small patch repair.

Epigenetic regulation of the MGMT and hMSH6 DNA repair genes in cells resistant to methylating agents.

We investigated the relationship between DNA cytosine methylation and the expression of two genes associated with resistance to DNA methylation damage. Variants of RajiMex- cells acquired resistance to N-methyl-N-nitrosourea by either reactivating a previously silent O6-methylguanine-DNA methyltransferase (MGMT) gene or by repressing the hMSH6 mismatch repair gene. DNA sequencing and measurements of mRNA and enzyme levels revealed that MGMT activity was not correlated with methylation of the core MGMT promoter. Treatment with the demethylating agent 5-azadeoxycytidine reduced MGMT mRNA and enzyme levels, indicating that methylation of some nonpromoter sequences may be required for MGMT gene expression. In contrast, both hMSH6 mRNA and protein levels were increased by 5-azadeoxycytidine treatment of an N-methyl-N-nitrosourea-resistant variant that did not express detectable hMSH6, which implies that this gene was transcriptionally silenced by cytosine methylation. This could be substantiated by in vitro modification of the CpG sites in the hMSH6 promoter with restriction methylase M.SssI, which abolished the transcription of a reporter gene under its control in a transient transfection assay. Taken together, our data show that treatment with chemical methylating agents alters gene expression patterns through increased CpG methylation of genomic DNA, and thereby permits the emergence and selection of clones that are resistant to these agents due to increased repair or tolerance of O6-methylguanine.

Coregulation of the human O6-methylguanine-DNA methyltransferase with two unrelated genes that are closely linked.

The loss of expression of the enzyme O6-methylguanine-DNA methyltransferase (the Mex- phenotype), which often results from cellular transformation, confers hypersensitivity to alkylating agents. We have observed two unrelated examples in which human cell lines have undergone a spontaneous alteration in their Mex phenotype during propagation in vitro. The change was reversible and was not the result of mutation. In both cases a loss of methyltransferase expression was accompanied by a simultaneous loss of expression of two metabolically unrelated enzymes: thymidine kinase and galactokinase. "Reversion" to methyltransferase expression was accompanied by simultaneous reexpression of both kinase activities. A third example of this coordinate gene regulation was seen with the Burkitt's lymphoma cell line Raji which expresses methyltransferase, thymidine kinase, and galactokinase at high levels. A thymidine kinase- Raji cell line derived by bromodeoxyuridine mutagenesis that is also Mex- was found to be galactokinase-. It appears that methyltransferase expression may in some instances be coordinately regulated with the tk and glk loci which are closely linked on human chromosome 17.

Spontaneous development of drug resistance: mismatch repair and p53 defects in resistance to cisplatin in human tumor cells.

The contributions of defective mismatch repair and mutated p53 to cisplatin resistance of human tumor cells were analysed. Mismatch repair defects were not associated with a predictable degree of resistance among several tumor cell lines. Repair defective variants of the A2780 ovarian carcinoma cell line which were isolated by selection for a methylation tolerant phenotype and did not express the hMLH1 mismatch repair protein, were highly resistant to cisplatin. Their cisplatin resistance was not a simple consequence of the mismatch repair defect. They were members of a drug-naive subpopulation of A2780 in which a silent hMLH1 gene accompanies a mutated p53. Two complementary approaches indicated that each defect contributes to cisplatin resistance independently and to a different extent. Firstly, separate introduction of a p53 defect into A2780 cells significantly increased their cisplatin resistance; defective hMLH1 provided less extensive protection. Secondly, azadeoxycytidine reactivation of the silent hMLH1 gene or expression of a transfected hMLH1 cDNA sensitized the doubly hMLH1/p53 deficient cells only slightly to cisplatin. Both approaches indicate that defective p53 status is a major determinant of cisplatin resistance and defective mismatch repair is a minor, and independent, contributor. The data have implications for the development of intrinsic cisplatin resistance.