Reto Brem

Reactive oxygen species generated by thiopurine/UVA cause irreparable transcription-blocking DNA lesions

Long-term treatment with the anticancer and immunosuppressant thiopurines, azathioprine or 6-mercaptopurine, is associated with acute skin sensitivity to ultraviolet A (UVA) radiation and a high risk of skin cancer. 6-thioguanine (6-TG) that accumulates in the DNA of thiopurine-treated patients interacts with UVA to generate reactive oxygen species. These cause lethal and mutagenic DNA damage. Here we show that the UVA/DNA 6-TG interaction rapidly, and essentially irreversibly, inhibits transcription in cultured human cells and provokes polyubiquitylation of the major subunit of RNA polymerase II (RNAPII). In vitro, 6-TG photoproducts, including the previously characterized guanine-6-sulfonate, in the transcribed DNA strand, are potent blocks to RNAPII transcription whereas 6-TG is only slightly inhibitory. In vivo, guanine-6-sulfonate is removed poorly from DNA and persists to a similar extent in the DNA of nucleotide excision repair-proficient and defective cells. Furthermore, transcription coupled repair-deficient Cockayne syndrome cells are not hypersensitive to UVA/6-TG, indicating that potentially lethal photoproducts are not selectively excised from transcribed DNA. Since persistent transcription-blocking DNA lesions are associated with acute skin responses to sunlight and the development of skin cancer, our findings have implications for skin cancer in patients undergoing thiopurine therapy.

Multiple forms of DNA Damage Caused by UVA Photoactivation of DNA 6-Thioguanine

Thiopurines are prescribed frequently as medication for cancer and for inflammatory disorders. One of them, azathioprine, has been the immunosuppressant of choice for organ transplant recipients for many years. Thiopurine use is associated with elevated sun sensitivity and skin cancer risk. Skin sensitization is selective for UVA. 6‐TG integrates into DNA and unlike the canonical DNA bases, it is a strong UVA chromophore with an absorbance maximum at 342 nm. DNA 6‐TG is a photosensitizer and a source of reactive oxygen species. Reactive oxygen that is generated from the photochemical activation of DNA 6‐TG causes extensive damage to DNA and proteins. This damage is mutagenic and extremely toxic to cultured human cells. Here we describe some of the lesions that are known to be generated from UVA irradiation of DNA 6‐TG. We discuss how this photochemical damage might contribute to the toxic effect of thiopurine/UVA treatment on cultured cells and to the high risk of skin cancer in thiopurine‐treated patients.

Efficient DNA interstrand crosslinking by 6-thioguanine and UVA radiation.

Patients taking the immunosuppressant and anticancer thiopurines 6-mercaptopurine, azathioprine or 6-thioguanine (6-TG), develop skin cancer at a very high frequency. Their DNA contains 6-TG which absorbs ultraviolet A (UVA) radiation, and their skin is UVA hypersensitive, consistent with the formation of DNA photodamage. Here we demonstrate that UVA irradiation of 6-TG-containing DNA causes DNA interstrand crosslinking. In synthetic duplex oligodeoxynucleotides, the interstrand crosslinks (ICLs) can form between closely opposed 6-TG bases and, in a less favoured reaction, between 6-TG and normal bases on the opposite strand. In vivo, UVA irradiation of cultured cells containing 6-TG-substituted DNA also causes ICL formation and induces the chromosome aberrations that are characteristically associated with this type of DNA lesion. 6-TG/UVA activates the Fanconi anemia (FA) pathway via monoubiquitination of the FANCD2 protein. Cells defective in the FA pathway or other factors involved in ICL processing, such as XPF and DNA Polζ, are all hypersensitive to killing by 6-TG/UVA-consistent with a significant contribution of photochemical ICLs to the cytotoxicity of this treatment. Our findings suggest that sunlight-exposed skin of thiopurine treated patients may experience chronic photochemical DNA damage that requires constant intervention of the FA pathway.

DNA breakage and cell cycle checkpoint abrogation induced by a therapeutic thiopurine and UVA radiation

The frequency of squamous cell skin carcinoma in organ transplant patients is around 100-fold higher than normal. This dramatic example of therapy-related cancer reflects exposure to sunlight and to immunosuppressive drugs. Here, we show that the interaction between low doses of UVA, the major ultraviolet component of incident sunlight, and 6-TG, a UVA chromophore that is introduced into DNA by one of the most widely prescribed immunosuppressive drugs, causes DNA single- and double-strand breaks (DSB). S phase cells are particularly vulnerable to this DNA breakage and cells defective in rejoining of S-phase DSB are hypersensitive to the combination of low-dose UVA and DNA 6-TG. 6-TG/UVA-induced DNA lesions provoke canonical DNA damage responses involving activation of the ATM/Chk2 and ATR/Chk1 pathways and appropriate cell cycle checkpoints. Higher levels of photochemical DNA damage induce a proteasome-mediated degradation of Chk1 and checkpoint abrogation that is consistent with persistent unrepaired DNA damage. These findings indicate that the interaction between UVA and an immunosuppressant drug causes photochemical DNA lesions, including DNA breaks, and can compromise cell cycle checkpoints. These two properties could contribute to the high risk of sunlight-related skin cancer in long-term immunosuppressed patients.

Global analysis of differential gene expression after transformation with the v-H-ras oncogene in a murine tumor model

Mouse PB-3c mast cells stably transfected with the v-H-ras oncogene induce tumor formation in vivo when implanted into mice. Such tumor cells are characterized by an autocrine IL-3 loop. DNA microarrays allow simultaneous transcript imaging of several thousand genes and the technique was applied in this tumor model to analyse gene expression following malignant transformation. Using three independent tumor lines derived from the same precursor the expression of about 400 out of 11 000 genes was modulated in each tumor. A subset of only 75 genes (0.68%) is shared and up- or downregulated in all three lines. A significant portion of this gene pool possesses functions related to tumorigenesis such as cell adhesion, signaling or transcriptional regulation. Apart from a number of expressed sequence tags (ESTs) we find downregulation of four interferon-inducible genes in the tumor lines. Finally, when we extrapolate our data to the complete mouse genome, we estimate that about 500 genes are differentially expressed in tumor cells compared to the precursor cell PB-3c.

Protein Oxidation and DNA Repair Inhibition by 6-Thioguanine and UVA Radiation

Damage to skin DNA by solar UV is largely unavoidable, and an optimal cellular response to it requires the coordinated operation of proteins in numerous pathways. A fully functional DNA repair proteome for removing harmful DNA lesions is a prerequisite for an appropriate DNA damage response. Genetically determined failure to repair UV-induced DNA damage is associated with skin photosensitivity and increased skin cancer risk. Patients treated with immunosuppressant/anti-inflammatory thiopurines are also photosensitive and have high rates of sun-related skin cancer. Their DNA contains the base analog 6-thioguanine (6-TG), which acts as a UVA photosensitizer to generate reactive oxygen species (ROS), predominantly singlet oxygen ((1)O2). ROS damage both DNA and proteins. Here we show that UVA irradiation of cultured human cells containing DNA 6-TG causes significant protein oxidation and damages components of the DNA repair proteome, including the Ku, OGG-1, MYH, and RPA proteins. Assays of DNA repair in intact cells or in cell extracts indicate that this protein damage compromises DNA break rejoining and base and nucleotide excision repair. As these experimental conditions simulate those in the skin of patients taking thiopurines, our findings suggest a mechanism whereby UVA in sunlight may contribute to skin carcinogenesis in immunosuppressed patients.

Oxidation-Mediated DNA Cross-Linking Contributes to the Toxicity of 6-Thioguanine in Human Cells

The thiopurines azathioprine and 6-mercaptopurine have been extensively prescribed as immunosuppressant and anticancer agents for several decades. A third member of the thiopurine family, 6-thioguanine (6-TG), has been used less widely. Although known to be partly dependent on DNA mismatch repair (MMR), the cytotoxicity of 6-TG remains incompletely understood. Here, we describe a novel MMR-independent pathway of 6-TG toxicity. Cell killing depended on two properties of 6-TG: its incorporation into DNA and its ability to act as a source of reactive oxygen species (ROS). ROS targeted DNA 6-TG to generate potentially lethal replication-arresting DNA lesions including interstrand cross-links. These triggered processing by the Fanconi anemia and homologous recombination DNA repair pathways. Allopurinol protected against 6-TG toxicity by acting as a ROS scavenger and preventing DNA damage. Together, our findings provide mechanistic evidence to support the proposed use of thiopurines to treat HR-defective tumors and for the coadministration of 6-TG and allopurinol as an immunomodulation strategy in inflammatory disorders.

6-Thioguanine damages mitochondrial DNA and causes mitochondrial dysfunction in human cells

The anticancer and immunosuppressant thiopurines cause 6‐thioguanine (6‐TG) to accumulate in nuclear DNA. We report that 6‐TG is also readily incorporated into mitochondrial DNA (mtDNA) where it is rapidly oxidized. The oxidized forms of mtDNA 6‐TG inhibit replication by DNA Pol‐γ. Accumulation of oxidized 6‐TG is associated with reduced mtDNA transcription, a decline in mitochondrial protein levels, and loss of mitochondrial function. Ultraviolet A radiation (UVA) also oxidizes mtDNA 6‐TG. Cells without mtDNA are less sensitive to killing by a combination of 6‐TG and UVA than their mtDNA‐containing counterparts, indicating that photochemical mtDNA 6‐TG oxidation contributes to 6‐TG‐mediated UVA photosensitization.

UVA photoactivation of DNA containing halogenated thiopyrimidines induces cytotoxic DNA lesions

Highlights • Growing cells incorporate thio-iodo-deoxyuridine and thio-bromo-deoxyuridine into DNA.• They are non-toxic but act as powerful UVA photosensitisers.• UVA lesions include DNA-protein and DNA–DNA crosslinks.• Singlet oxygen is involved in the formation of this potentially lethal damage.• Thio-halo-deoxynucleosides offer a potential selective therapeutic option.

DNA repair inhibition by UVA photoactivated fluoroquinolones and vemurafenib

Cutaneous photosensitization is a common side effect of drug treatment and can be associated with an increased skin cancer risk. The immunosuppressant azathioprine, the fluoroquinolone antibiotics and vemurafenib—a BRAF inhibitor used to treat metastatic melanoma—are all recognized clinical photosensitizers. We have compared the effects of UVA radiation on cultured human cells treated with 6-thioguanine (6-TG, a DNA-embedded azathioprine surrogate), the fluoroquinolones ciprofloxacin and ofloxacin and vemurafenib. Despite widely different structures and modes of action, each of these drugs potentiated UVA cytotoxicity. UVA photoactivation of 6-TG, ciprofloxacin and ofloxacin was associated with the generation of singlet oxygen that caused extensive protein oxidation. In particular, these treatments were associated with damage to DNA repair proteins that reduced the efficiency of nucleotide excision repair. Although vemurafenib was also highly phototoxic to cultured cells, its effects were less dependent on singlet oxygen. Highly toxic combinations of vemurafenib and UVA caused little protein carbonylation but were nevertheless inhibitory to nucleotide excision repair. Thus, for three different classes of drugs, photosensitization by at least two distinct mechanisms is associated with reduced protection against potentially mutagenic and carcinogenic DNA damage.