Thomas M. Rünger

Impaired S-phase transit of Werner syndrome cells expressed in lymphoblastoid cell lines☆

The clinical phenotype of Werners syndrome (WS) includes short stature, premature cataracts, skin atrophy, osteoporosis, graying and loss of hair, neoplasia, diabetes mellitus, and arteriosclerosis. Cultured cells from patients with this autosomal recessive disorder exhibit chromosomal instability and a markedly reduced replicative lifespan and growth rate. To elucidate the cell cycle alterations associated with the growth deficit, we continuously labeled lymphoid cell lines from five WS patients and from four healthy adult controls with 5-bromodeoxyuridine. Bivariate Hoechst 33258/ethidium bromide flow cytometry revealed a 2.4-h prolongation in the minimal duration of the S phase of WS cells (P less than 0.005). Moreover, the fraction of proliferating cells irreversibly arrested in the S phase (5.4% vs 1.4% in controls) was significantly elevated in WS (P less than 0.001). Other cell cycle compartments were not significantly affected in WS cell lines. As a partial test of the hypothesis that the WS phenotype is due to a defect in DNA topoisomerase I (topo I) or DNA topoisomerase II (topo II) we exposed lymphoid cells from a healthy control to the topo I inhibitor camptothecin or to the topo II inhibitor 4-(9-acridinylamino)methanesulfon-m-anisidine. The cell kinetic alterations elicited by these compounds differed from that exhibited by untreated WS patients. Thus, a primary defect in topo I or II is unlikely in WS. Our cell cycle results, however, provide important evidence that the biochemical genetic lesion is in fact expressed in lymphoblastoid cell lines, the most readily available cells from such subjects.

ASSESSMENT OF DNA DAMAGE INDUCED BY BROADBAND AND NARROWBAND UVB IN CULTURED LYMPHOBLASTS AND KERATINOCYTES USING THE COMET ASSAY

Phototherapy with broadband UVB is an effective treatment for inflammatory dermatoses. A newly developed fluorescent UVB lamp (Philips TL01) that emits a narrowband UVB around 311 nm was shown to be superior for the phototherapy of psoriasis. In order to contribute to the knowledge about the carcinogenic potential of this UVB source, we measured the DNA damage in lymphoblasts and keratinocytes induced by narrowband UVB and compared it with that by conventional broadband UVB using the single cell gel electrophoresis (comet as‐say). At equal doses, broadband UVB produced more DNA damage than narrowband UVB. However, in phototherapy of psoriasis, up to 10‐fold higher doses are used with TLO1. When therapeutically equivalent doses were compared (10‐fold correction for narrowband UVB), we found only slight differences in the amount of DNA damage produced by broadband and narrowband UVB. This supports the already existing evidence that for phototherapy narrowband UVB is not more carcinogenic than broadband UVB.

Genotoxicity and mutagenicity of the α,β-unsaturated carbonyl compound crotonaldehyde (butenal) on a plasmid shuttle vector

Crotonaldehyde is an α,β-unsaturated carbonyl compound and an important environmental and industrial toxic substance. Its mutagenic and carcinogenic properties are related to its reactivity to DNA, where it forms different guanine adducts. In order to study the mutagenic consequences of this agent in intact human cells, we treated the shuttle vector plasmid pZ189 with different doses of crotonaldehyde at 37°C for 2 h and then transfected the such damaged plasmid into the normal human lymphoblast cell line GM0621. Within these host cells the guanine adducts are repaired and the plasmids replicated by cellular enzymes. After 2.5 days replicated plasmids were purified from the cells and plasmid survival was quantitated by transformation ability. With increasing doses of crotonaldehyde, we found a significant decline of plasmid survival, reflecting a pronounced genotoxicity of crotonaldehyde-induced DNA damage in intact human cells. Using the plasmid encoded mutagenesis marker gene supF, we were able to screen for mutants and determine mutation frequency in recovered plasmids. A significant increase in mutation frequency with increasing doses of crotonaldehyde reflects mutagenicity of crotonaldehyde-induced DNA damage. Base sequence analysis of recovered mutants revealed 39% point mutations, 46% deletions, and 15% insertions and inversions. Most of the point mutations (82%) were located at G:C base pairs, which is well explained by the DNA damage profile of crotonaldehyde. Among deletions we found a frequent reoccurrence of two hot spot deletions, representing 62% of all deletions. The sites of breakpoints of these deletions hot spots and of other deletions within the plasmid were also found to be sites of DNA breaks, directly induced by crotonaldehyde, as seen in an endlabeled plasmid fragment, treated with crotonaldehyde. Further analysis of the flanking sequences around the deletion breakpoints revealed a high frequency of four different kinds of short sequence homologies of up to eight base pairs.

Processing of Directly and Indirectly Ultraviolet-Induced DNA Damage in Human Cells

Mutations caused by ultraviolet (UV)-induced DNA damage represent the initial genetic changes in the tumorigenesis of UV-induced skin cancer. Different wavelengths of UV radiation cause different kinds of DNA damage and mutations. UVB (290-320 nm) generates pyrimidine dimers by direct excitation of the DNA molecule. UVA (320-400 nm) can damage the DNA only indirectly through a photosensitized reaction. This indirect action is mediated mainly by singlet oxygen, which generates purine base modifications, and has been implicated in the carcinogenic effects of UVA. In order to study the processing of directly and indirectly UV-induced DNA damage in human cells, we first treated the replicating plasmid pRSVcat with up to 10 kJ/m2 UVB or with the photosensitizer methylene blue plus visible light (which generates singlet oxygen) in vitro. Then, the damaged plasmid was transfected into normal or repair deficient xeroderma pigmentosum complementation group A (XP-A) cells. DNA repair was assessed by measuring activity of reactivated chloramphenicol acetyltransferase (CAT) enzyme, encoded by the plasmids cat gene, in cell extracts after 3 days. While XP-A cells exhibited a significantly reduced repair of UVB-induced DNA damage, they showed a normal repair of singlet oxygen-induced DNA damage. This indicates a differential DNA repair pathway for directly and indirectly UV-induced DNA damage in human cells. Irradiation of the plasmid with UVA alone did not result in a genotoxic effect. Only in conjunction with a cell extract, which provides all candidate cellular photosensitizers, did we find a reduced CAT activity after transfection. This indicates that the genotoxicity of UVA is mediated by a cellular photosensitizer.

Specific Autologous Anti-Melanoma T Cell Response in vitro Using Monocyte-Derived Dendritic Cells

Dendritic cells (DC) are antigen-presenting cells initiating primary and secondary immune responses. Since malignant tumors are able to escape immunologic control, DC might be prime candidates to activate the immune system against tumor cells. In an autologous system, a dynamic interaction among monocyte-derived DC (MoDC), T lymphocytes, and tumor cells obtained from melanoma patients could be noted. MoDC were generated from blood monocytes in the presence of GM-CSF, IL-4, and IFN-gamma. T cells were isolated either from peripheral blood or from lymph nodes. Melanoma cells were harvested from surgically removed tumor metastases. They were then gamma-irradiated and co-cultured with autologous MoDC and T lymphocytes. After 5 days, the lymphocytes showed a high proliferative activity and the majority of them were CD8-positive. In five cases tested, they revealed a high cytotoxic activity resulting in apoptosis of tumor cells. These findings suggest that MoDC are capable of initiating an effective specific anti-tumor response in a strictly autologous mixed lymphocyte tumor culture (MLTC), even though tumor-specific antigens had not been individually defined. Therefore (I) whole melanoma cells can serve as a source of antigen, (II) monocyte-derived dendritic cells may process and present melanoma-specific antigens resulting in a strong lymphocyte proliferation, (III) the majority of responding T lymphocytes are CD8-positive, and (IV) an acquired cytotoxic response eventually leads to apoptosis of the melanoma cells. The reaction demonstrated here permits to in vitro and quantitatively monitoring the effect of T cell directed immunotherapies such as the adoptive immunotherapy of tumors.

Atypical varicella-zoster virus infection in an immunocompromised patient: Result of a virus-induced vasculitis

We describe a patient with cutaneous T-cell lymphoma in whom persistent, painless, ecthymatous nodules developed as a result of a varicella-zoster virus infection. The localized infection occurred without a vesicular stage. Ultrastructural studies revealed a lack of epidermal involvement and massive varicella-zoster virus replication within endothelial cells, leading to an obliterative vasculitis. This suggests direct infection of dermal vessels from adjacent nerves, bypassing the epidermis, which is usually infected first in the classic infectious pathway during varicella-zoster virus reactivation from sensory nerves.

DNA damage formation, DNA repair, and survival after exposure of DNA repair-proficient and nucleotide excision repair-deficient human lymphoblasts to UVA1 and UVB

Purpose : The comet assay has been used to visualize DNA damage in single cells after exposure to UV light. These comets are commonly thought to reflect transient, repair-induced DNA breaks. The goal of the work presented here was to further characterize the nature of UV-induced comets and to further elucidate DNA damage formation by different wavelengths of ultraviolet light. Materials and methods : Detailed dose–response and time-course experiments with comet formation were carried out with normal and nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) lymphoblasts. Irradiation was carried out with low, intermediate, or high doses of UVA1 or UVB, comet formation was observed, cell survival and viability were determined, and UV-induced apoptosis was measured. Results : All responses were dose-dependent. With the intermediate dose of UVA1, a pronounced comet formation was observed without subsequent growth inhibition. Raising levels of porphyrins, which act as photosensitizers, by preincubation with 5-amino-levulinic acid increased comet formation with UVA1, but not with UVB. UVA1-sensitivity and comet formation in XP cells was not significantly different from the normal cells. With UVB no comet formation was seen without subsequent apoptotic cell death. XP cells exhibited the known UVB-hypersensitivity, but their comet formation was not significantly different from that of normal cells. Conclusions : The findings are compatible with the hypothesis that UV-induced comets represent transient repair-induced DNA breaks. Both, the NER of dimers and the base excision repair of oxidative DNA modifications are thought to contribute to comet formation.PURPOSEnThe comet assay has been used to visualize DNA damage in single cells after exposure to UV light. These comets are commonly thought to reflect transient, repair-induced DNA breaks. The goal of the work presented here was to further characterize the nature of UV-induced comets and to further elucidate DNA damage formation by different wavelengths of ultraviolet light.nnnMATERIALS AND METHODSnDetailed dose-response and time-course experiments with comet formation were carried out with normal and nucleotide excision repair (NER)-deficient xeroderma pigmentosum (XP) lymphoblasts. Irradiation was carried out with low, intermediate, or high doses of UVA1 or UVB, comet formation was observed, cell survival and viability were determined, and UV-induced apoptosis was measured.nnnRESULTSnAll responses were dose-dependent. With the intermediate dose of UVA1, a pronounced comet formation was observed without subsequent growth inhibition. Raising levels of porphyrins, which act as photosensitizers, by preincubation with 5-amino-levulinic acid increased comet formation with UVA1, but not with UVB. UVA1-sensitivity and comet formation in XP cells was not significantly different from the normal cells. With UVB no comet formation was seen without subsequent apoptotic cell death. XP cells exhibited the known UVB-hypersensitivity, but their comet formation was not significantly different from that of normal cells.nnnCONCLUSIONSnThe findings are compatible with the hypothesis that UV-induced comets represent transient repair-induced DNA breaks. Both, the NER of dimers and the base excision repair of oxidative DNA modifications are thought to contribute to comet formation.

A novel plasmid shuttle vector for the detection and analysis of microsatellite instability in cell lines

Microsatellite instability is an important feature of tumors from hereditary nonpolyposis colorectal carcinoma (HNPCC) patients as well as a variety of sporadic tumors. Here, we present a novel plasmid shuttle vector for the detection of this replication error (RER+) phenotype in human cell lines. The episomely replicated plasmid pZCA29 harbours the bacterial beta-galactosidase gene interrupted by two palindromically arranged poly-(CA)-repeat tracts. The resulting + 1-frameshift leads to white colonies of Escherichia coli DH10B on X-Gal/IPTG1 agar plates. Mutations in the repeats characteristic of the RER+-phenotype may result in the loss or gain of CA-repeats leading to blue bacterial colonies. We transiently transfected the colorectal cancer cell lines SW480 and HCT116 with the plasmid pZCA29, isolated replicated plasmid DNA after several days and used it to transform E. coli DH10B. We found 1.0 to 1.7% blue colonies after passage of the plasmid through the RER+-cell line SW480 in contrast to 3.5 to 8.1% blue colonies after transfection of the RER+-cell line HCT116, the mutation frequencies increasing with incubation time. Sequence analysis of mutated plasmids revealed mostly 2-bp deletions which occurred especially in one of the repeat tracts. We conclude that pZCA29 appears to be a suitable shuttle vector for the detection and analysis of a RER+-phenotype in cell lines.

Erythrokeratodermia progressiva symmetrica Darier-Gottron mit generalisierter Ausprägung

ZusammenfassungWir berichten über Mutter und Sohn mit Erythrokeratodermia progressiva symmetrica Darier-Gottron. Beide Patienten entwickelten im Alter von 6 Monaten symmetrische erythematosquamöse Plaques an den Extremitäten und im Gesicht. Beim Sohn kam es mit 2u20051/2 Jahren zu einer raschen Ausbreitung mit Befall des gesamten Integuments. Die Mutter berichtet über einen ähnlichen Verlauf, jedoch mit spontaner Regression ab dem 10. Lebensjahr. Die Klinik dieses generalisierten Zustandes war identisch mit dem Befund einer kongenitalen lamellären Ichthyose. Lichtmikroskopisch ergaben sich unspezifische Veränderungen mit Orthohyperkeratose, fokalen Parakeratosearealen und Akanthose. Elektronenmikroskopisch fanden sich im Stratum granulosum eine hohe Keratinosomenanzahl, Keratinosomenlamellen in den Interzellularräumen und teils vermehrtes Keratohyalin mit Verklumpung. Weiterhin waren kurze Tonofilamentbündel mit Verklumpungen im Stratum spinosum auffällig. Systemische Retinoide brachten dem Sohn eine mehrere Monate anhaltende Befundbesserung. Die Mutter berichtet über ähnlich gute Erfolge unter einer Retinoidintervalltherapie. Die Beobachtung zeigt die Schwierigkeit, die Erythrokeratodermia progressiva symmetrica als eigentlich lokalisierte Verhornungsstörung bei jedoch zwischenzeitlich ausgedehntem, generalisiertem Zustand mit den derzeitig zur Verfügung stehenden klinischen und ultrastrukturellen Kriterien zuverlässig von anderen Verhornungsstörungen abzugrenzen.SummaryA mother and her son presented with erythrokeratodermia progressiva symmetrica Darier-Gottron. Both patients developed symmetrical erythematous and hyperkeratotic plaques on the extremities and face at the age of 6 months. At the age of 2u20051/2 years the son suffered from rapid progression of the disease to involve the entire skin. The disesase of his mother had shown a similar course, however, with spontaneous regression at the age of 10 years. The clinical features of this generalized condition were identical to congenital lamellar ichthyosis. Light microscopy was non-specific with orthohyperkeratosis, focal parakeratosis and acanthosis. Electron microscopy revealed numerous keratinosomes in the stratum granulosum, keratinosome-derived lamellae in the intercellular space and partly augmented keratohyalin with clumping. In the stratum spinosum short tonofilament bundles with clumping were remarkable. The child experienced a significant and persistent improvement with systemic retinoids. His mother´s disease was successfully controlled with intermittent retinoid therapy. With the clinical and ultrastructural criteria presently available, an unambiguous differentiation between erythrokeratodermia progressiva symmetrica, usually a localized disorder of keratinization, however with intermittent generalization, and other disorders of keratinization seems difficult.

Exploring the role of oxygen in Fanconi's anemia.

Fanconi’s anemia (FA) is a clinically and genetically heterogeneous disease that presents with a wide spectrum of clinical manifestations, ranging from severe congenital malformations to a completely normal phenotype (Auerbach et al. 1989). The onset of progressive bone marrow failure may occur in early childhood or as late as at 40 years of age. The cellular phenotype of FA consists of chromosomal instability, a cell cycle defect, and increased sensitivity to certain clastogens, as well as oxygen (Hoehn et al. 1989). In analogy to what has recently become known about the embryo-protective role of the p53 gene product (Nicol et al. 1995), leaky protection against endogenous free radical-mediated DNA damage could explain both the variable pattern of developmental abnormalities and the variable onset of bone marrow failure in Fanconi’s anemia. Cell culture studies have found no evidence for defective free radical scavenger systems (Gille et al. 1987; Joenje and Gille 1989), but they have also shown that in FA cells, chromosomal breakage and cell growth are influenced strongly by oxygen (Joenje et al. 1981; Schindler and Hoehn 1988). In this chapter we review a number of approaches by which we hope to clarify the pathogenetic role of oxygen in Fanconi’s anemia.