Christiane Kuschal

Lessons learned from DNA repair defective syndromes

Abstract:u2002 Genomic instability is the driving force behind cancer development. Human syndromes with DNA repair deficiencies comprise unique opportunities to study the clinical consequences of faulty genome maintenance leading to premature aging and premature cancer development. These syndromes include chromosomal breakage syndromes with defects in DNA damage signal transduction and double‐strand break repair, mismatch repair defective syndromes as well as nucleotide excision repair defective syndromes. The same genes that are severely affected in these model diseases may harbour more subtle variations in the ‘healthy’ normal population leading to genomic instability, cancer development, and accelerated aging at later stages of life. Thus, studying those syndromes and the molecular mechanisms behind can significantly contribute to our understanding of (skin) cancerogenesis as well as to the development of novel individualized preventive and therapeutic anticancer strategies. The establishment of centers of excellence for studying rare genetic model diseases may be helpful in this direction.

Skin cancer in organ transplant recipients: effects of immunosuppressive medications on DNA repair

Abstract:u2002 UV‐induced skin cancers comprise a major problem in organ transplant recipients (OTRs). Cyclosporin A, a calcineurin inhibitor, is used as a standard immunosuppressant and clearly increases the skin cancer risk. Azathioprine does not appear to result in such an increase in skin cancer risk, and mTOR inhibitors are associated with an even lesser skin cancer risk. The underlying molecular mechanisms of these clinically important differences among immunosuppressants are still unclear and may relate to other than immunological effects. Insights may be gained by the multistep skin cancer theory and xeroderma pigmentosum, where defective nucleotide excision repair (NER) results in a cellular mutator phenotype and cutaneous carcinogenesis. This viewpoint assay summarizes current knowledge about the influence of the most commonly used immunosuppressive drugs in OTRs on DNA repair. Calcineurin inhibition results in a 200‐fold increased skin cancer risk compared with the normal population and inhibits NER. The skin cancer risk under azathioprine is threefold less compared with calcineurin inhibitors, which may relate to inhibition of only the last step of NER, i.e. gap filling. mTOR inhibitors do not reduce NER in the global genome and can inhibit the growth of already initiated tumors, which may account for the markedly reduced skin cancer risk compared with calcineurin inhibitors. We conclude that OTRs may benefit from treatment regimens other than calcineurin inhibitors and speculate that a targeted modulation of calcineurin‐dependent signalling may prevent UV‐induced tumor formation by enhancing NER not only in OTRs but also in the general population, at least in part.

Cyclosporin A, but not everolimus, inhibits DNA repair mediated by calcineurin: implications for tumorigenesis under immunosuppression

Abstract:u2002 Unlike other immunosuppressive drugs including everolimus, cyclosporin A causes a dramatic increase of UV‐induced skin cancer, a feature that is reminiscent of xeroderma pigmentosum (XP), where defective nucleotide excision repair (NER) of UV‐induced DNA damage results in cutaneous carcinogenesis. The molecular basis of the clinically important differential activities of cyclosporin A and everolimus is still unclear. We measured post‐UV cell survival of cyclosporin A‐ and everolimus‐treated human fibroblasts and lymphoblasts using a cell proliferation assay (MTT). The cellular NER capacity was assessed by host cell reactivation. Using an ELISA and specific antibodies, cyclobutane pyrimidine and pyrimidine‐6,4‐pyrimidone photoproduct removal from the cellular genome was measured. The effect of calcineurin on NER was investigated using a calcineurin A expression vector and specific RNAi. Cyclosporin A led to a dose dependent decrease in post‐UV cell survival, inhibited NER and blocked photoproduct removal. In contrast, none of these effects where seen in everolimus‐treated cells. Overexpression of calcineurin A resulted in increased NER and complemented the Cyclosporin A‐induced reduction of NER. Downregulation of calcineurin using RNAi inhibited NER comparable to cyclosporin A‐treatment. We conclude that cyclosporin A, but not everolimus, leads to an increased skin cancer risk via a calcineurin signalling‐dependent impairment of NER.

Repair of UV photolesions in xeroderma pigmentosum group C cells induced by translational readthrough of premature termination codons

Significance About 12% of human genetic disorders involve premature stop codons (PTC) that may produce abnormal, short proteins. Some antibiotics and other compounds have been proposed to restore full-length proteins by reading through PTC. We studied skin cells from xeroderma pigmentosum (XP) patients with different PTC in a DNA repair gene. XP patients have a DNA repair defect and a 10,000-fold increased risk of sunlight-induced skin cancer. Using several readthrough compounds, we found increased levels of DNA repair protein, assembly of DNA repair proteins at the DNA damage site, and repair of UV damage in some XP cells. Even small amounts of increased DNA repair protein may provide potential therapy for XP patients and may reduce disease severity. About 12% of human genetic disorders involve premature termination codons (PTCs). Aminoglycoside antibiotics have been proposed for restoring full-length proteins by readthrough of PTC. To assess the efficiency of readthrough, we selected homozygous and compound heterozygous skin fibroblasts from xeroderma pigmentosum (XP) patients with different PTCs in the XPC DNA repair gene. XP patients have a nucleotide excision repair defect and a 10,000-fold increased risk of UV-induced skin cancer. In six of eight PTC-containing XP-C cells, treatment with Geneticin and gentamicin resulted in (i) stabilized XPC–mRNA, which would have been degraded by nonsense-mediated decay; (ii) increased expression of XPC protein that localized to UV-damaged sites; (iii) recruitment of XPB and XPD proteins to UV DNA damage sites; and (iv) increased repair of 6–4 photoproducts and cyclobutane pyrimidine dimers. Expression of PTC in a transfected vector revealed that readthrough depends on the PTC sequence and its location within the gene. This sensitive DNA repair assay system demonstrates the complexity of response to PTC readthrough inducers. The efficiency of aminoglycoside-mediated readthrough depends on the type and copy number of PTC, the downstream 4+ nucleotide, and the location within the exon. Treatment with small-molecule nonaminoglycoside compounds (PTC124, BZ16, or RTC14) resulted in similarly increased XPC mRNA expression and photoproduct removal with less toxicity than with the aminoglycosides. Characterizing PTC structure and parameters governing effective PTC readthrough may provide a unique prophylactic therapy for skin cancer prevention in XP-C patients.

Strict sun protection results in minimal skin changes in a patient with xeroderma pigmentosum and a novel c.2009delG mutation in XPD (ERCC2).

Abstract:u2002 We examined the clinical, molecular and genetic features of a 16‐year‐old boy (XP2GO) with xeroderma pigmentosum (XP) and progressive neurological symptoms. The parents are not consanguineous. Increased sun sensitivity led to the diagnosis of XP at 2u2003years of age and a strict UV protection scheme was implemented. Besides recurrent conjunctivitis and bilateral pterygium, only mild freckling was present on his lips. He shows absent deep tendon reflexes, progressive sensorineural deafness and progressive mental retardation. MRI shows diffuse frontal cerebral atrophy and dilated ventricles. Symptoms of trichothiodystrophy (brittle hair with a tiger‐tail banding pattern on polarized microscopy) or Cockayne syndrome (cachectic dwarfism, cataracts, pigmentary retinopathy and spasticity) were absent. XP2GO fibroblasts showed reduced post‐UV cell survival (D37u2003=u20033.8u2003J/m2), reduced nucleotide excision repair, reduced expression of XPD mRNA and an undetectable level of XPD protein. Mutational analysis of the XPD gene in XP2GO revealed two different mutations: a common p.Arg683Trp amino acid change (c.2047C>T) known to be associated with XP and a novel frameshift mutation c.2009delG (p.Gly670Alafs*39). The latter mutation potentially behaves as a null allele. While not preventing neurological degeneration, early diagnosis and rigorous sun protection can result in minimal skin disease without cancer in XP patients.

Cyclosporin A inhibits nucleotide excision repair via downregulation of the xeroderma pigmentosum group A and G proteins, which is mediated by calcineurin inhibition.

Abstract:u2002 Cyclosporin A (CsA) inhibits nucleotide excision repair (NER) in human cells, a process that contributes to the skin cancer proneness in organ transplant patients. We investigated the mechanisms of CsA‐induced NER reduction by assessing all xeroderma pigmentosum (XP) genes (XPA‐XPG). Western blot analyses revealed that XPA and XPG protein expression was reduced in normal human GM00637 fibroblasts exposed to 0.1 and 0.5u2003μm CsA. Interestingly, the CsA treatment reduced XPG, but not XPA, mRNA expression. Calcineurin knockdown in GM00637 fibroblasts using RNAi led to similar results suggesting that calcineurin‐dependent signalling is involved in XPA and XPG protein regulation. CsA‐induced reduction in NER could be complemented by the overexpression of either XPA or XPG protein. Likewise, XPA‐deficient fibroblasts with stable overexpression of XPA (XP2OS‐pCAH19WS) did not show the inhibitory effect of CsA on NER. In contrast, XPC‐deficient fibroblasts overexpressing XPC showed CsA‐reduced NER. Our data indicate that the CsA‐induced inhibition of NER is a result of downregulation of XPA and XPG protein in a calcineurin‐dependent manner.

Readthrough of stop codons by use of aminoglycosides in cells from xeroderma pigmentosum group C patients

Readthrough of premature termination (stop) codons (PTC) is a new approach to treatment of genetic diseases. We recently reported that readthrough of PTC in cells from some xeroderma pigmentosum complementation group C (XP‐C) patients could be achieved with the aminoglycosides geneticin or gentamicin. We found that the response depended on several factors including the PTC sequence, its location within the gene and the aminoglycoside used. Here, we extended these studies to investigate the effects of other aminoglycosides that are already on the market. We reasoned that topical treatment could deliver much higher concentrations of drug to the skin, the therapeutic target, and thus increase the therapeutic effect while reducing renal or ototoxicity in comparison with systemic treatment. Our prior clinical studies indicated that only a few percent of normal XPC expression was associated with mild clinical disease. We found minimal cell toxicity in the XP‐C cells with several aminoglycosides. We found increased XPC mRNA expression in PTC‐containing XP‐C cells with G418, paromomycin, neomycin and kanamycin and increased XPC protein expression with G418. We conclude that in selected patients with XP, topical PTC therapy can be investigated as a method of personalized medicine to alleviate their cutaneous symptoms.

Effect of DNA repair host factors on temozolomide or dacarbazine melanoma treatment in Caucasians

Objectives The efficacy of temozolomide (TMZ) or dacarbazine (DTIC) in melanoma treatment depends on low O-6-methylguanine-DNA-methyltransferase (MGMT) repair and on high mismatch repair. The aim of this study was to identify individual host markers for hematologic side effects and the treatment efficacy of TMZ or DTIC in melanoma treatment. Methods Fifty-one Caucasian patients with metastasized melanoma were recruited. In each patient, the mRNA expression of MGMT and two essential mismatch repair genes, MLH1 and MSH2, was measured in peripheral blood. The coding gene regions, including splice sites, were sequenced to identify genetic variants, and the promoter methylation status of the genes was determined. Results Both constitutively low and high mRNA expression of MGMT, MLH1, and MSH2 were significantly associated with reduced hematologic side effects (Pu2009=u20090.008–0.020), but did not correlate with treatment efficacy. We identified five variants in the MGMT gene, 13 variants in MLH1, and seven variants in MSH2, including five novel genetic variants in MLH1. Variations of the hosts gene expression of MGMT, MLH1, and MSH2 did not result from promoter methylation. Of note, one variant in MSH2 (rs2303428) was associated with increased hematologic side effects and showed a tendency for better treatment response. Conclusion Our results indicate that either low or high host expression of MGMT, MLH1, and MSH2 may serve as a marker for reduced hematologic side effects of TMZ or DTIC, but not for treatment efficacy in melanoma. The genetic variant rs2303428 (MSH2) might serve as a predictive marker for hematologic side effects and treatment response.

Temozolomide chemoresistance heterogeneity in melanoma with different treatment regimens: DNA damage accumulation contribution.

The efficacy of temozolomide in melanoma treatment is low (response rate <20%) and may depend on the activity of O6-methylguanine DNA methyltransferase (MGMT) and mismatch repair. We identified melanoma cell lines with different sensitivities to single versus prolonged clinical dosing regimens of temozolomide treatment and assessed a variety of potential resistance mechanisms using this model. We measured mRNA expression and promoter methylation of MGMT and essential mismatch repair genes (MLH1, MSH2). Cell cycle distribution, apoptosis/necrosis induction, O6-methylguanine-adduct formation, and ABCB1 gene expression were assessed. We found that three cell lines, MelA, MelB, and MelC, were more sensitive to a single dose regimen than to a prolonged regimen, which would be expected to exhibit higher cytotoxicity. KAII and LIBR cell sensitivity was higher with regard to the prolonged treatment regimen, as expected. Only MelC expressed MGMT. Gene expression correlated well with promoter methylation. Temozolomide exposure did not alter mRNA expression. Different sensitivities to temozolomide were caused neither by delayed apoptosis induction due to early cell cycle arrest nor by O6-methylguanine-adduct formation or efflux transporter expression. MelC was the most resistant cell line with rapid elimination of O6-methylguanine adducts. This was in good agreement with its MGMT expression. The sensitive cell lines KAII and LIBR accumulated O6-methylguanine adducts after a second treatment cycle with temozolomide in contrast with the other three cell lines. We conclude that MGMT expression and DNA adduct accumulation are relevant factors in temozolomide chemosensitivity. Considering individualized temozolomide treatment regimens either by quantification of DNA adducts or by chemosensitivity testing seems worthwhile clinically.

GTF2E2 Mutations Destabilize the General Transcription Factor Complex TFIIE in Individuals with DNA Repair-Proficient Trichothiodystrophy

The general transcription factor IIE (TFIIE) is essential for transcription initiation by RNA polymerase II (RNA pol II) via direct interaction with the basal transcription/DNA repair factor IIH (TFIIH). TFIIH harbors mutations in two rare genetic disorders, the cancer-prone xeroderma pigmentosum (XP) and the cancer-free, multisystem developmental disorder trichothiodystrophy (TTD). The phenotypic complexity resulting from mutations affecting TFIIH has been attributed to the nucleotide excision repair (NER) defect as well as to impaired transcription. Here, we report two unrelated children showing clinical features typical of TTD who harbor different homozygous missense mutations in GTF2E2 (c.448G>C [p.Ala150Pro] and c.559G>T [p.Asp187Tyr]) encoding the beta subunit of transcription factor IIE (TFIIEβ). Repair of ultraviolet-induced DNA damage was normal in the GTF2E2 mutated cells, indicating that TFIIE was not involved in NER. We found decreased protein levels of the two TFIIE subunits (TFIIEα and TFIIEβ) as well as decreased phosphorylation of TFIIEα in cells from both children. Interestingly, decreased phosphorylation of TFIIEα was also seen in TTD cells with mutations in ERCC2, which encodes the XPD subunit of TFIIH, but not in XP cells with ERCC2 mutations. Our findings support the theory that TTD is caused by transcriptional impairments that are distinct from the NER disorder XP.