Investigation of mechanisms involved in ultraviolet B radiation-induced, T cell -mediated immune suppression

Cutaneous exposure to ultraviolet-B radiation (UVR) results in the suppression of cell-mediated immune responses such as contact hypersensitivity (CHS) and delayed-type hypersensitivity (DTH). This modulation of immune responses is mediated by local or systemic mechanisms, both of which are associated with the generation of antigen-specific suppressor T lymphocytes (Ts). UV-induced Ts have been shown to be CD3+CD4+CD8 − T cells that control multiple immunological pathways. However, the precise mechanisms involved in the generation and function of these immunoregulatory cells remain unclear. We investigated the cellular basis for the generation of UV-induced Ts lymphocytes in both local and systemic models of immune suppression, and further examined the pleiotrophic function of these immunoregulatory cells. ^ We used Thy1.1 and Thy1.2 congenic mice in a draining lymph node (DLN) cell transfer model to analyze the role played by epidermal Langerhans cells in the generation of Ts cells. We demonstrate that T cells tightly adhered to antigen-presenting cells (APC) from UV-irradiated skin are the direct progenitors of UV-induced Ts lymphocytes. Our studies also reveal that UV-induced DNA-damage in the form of cyclobutyl pyrimidine dimers (CPD) in the epidermal APC is crucial for the altered maturation of these adherent T cells into Ts. ^ We used TCR transgenic mice in an adoptive transfer model and physically tracked the antigen-specific clones during immune responses in unirradiated versus UV-irradiated mice. We demonstrate that UV-induced Ts and effector TDTH cells share the same epitope specificity, indicating that both cell populations arise from the same clonal progenitors. UVR also causes profound changes in the localization and proliferation of antigen-specific T cells during an immune response. Antigen-specific T cells are not detectable in the DLNs of UV-irradiated mice after 3 days post-immunization, but are found in abundance in the spleen. In contrast, these clones continue to be found in the DLNs and spleens of normal animals several days post-immunization. Our studies also reveal that a Th2 cytokine environment is essential for the generation of Ts in UV-irradiated mice. ^ The third part of our study examined the pleiotrophic nature of UV-induced Ts. We used a model for the induction of both cellular and humoral responses to human gamma-globulin (HGG) to demonstrate that UV-induced Ts lymphocytes can suppress DTH as well as antibody responses. (Abstract shortened by UMI.) ^

Impact of BCR -ABL on DNA repair

The BCR-ABL fusion gene is the molecular hallmark of Philadelphia-positive leukemias. Normal Bcr is a multifunctional protein, originally localized to the cytoplasm. It has serine kinase activity and has been implicated in cellular signal transduction. Recently, it has been reported that Bcr can interact with xeroderma pigmentosum group B (XPB/ERCC3)—a nuclear protein active in UV-induced DNA repair. Two major Bcr proteins (p160 Bcr and p130Bcr) have been characterized, and our preliminary results using metabolic labeling and immunoblotting demonstrated that, while both the p160 and p130 forms of Bcr localized to the cytoplasm, the p130 form (and to a lesser extent p160) could also be found in the nucleus. Furthermore, electron microscopy confirmed the presence of Bcr in the nucleus and demonstrated that this protein associates with metaphase chromatin as well as condensed interphase heterochromatin. Since serine kinases that associate with condensed DNA are often cell cycle regulatory, these observations suggested a novel role for nuclear Bcr in cell cycle regulation and/or DNA repair. However, cell cycle synchronization analysis did not demonstrate changes in levels of Bcr throughout the cell cycle. Therefore we hypothesized that BCR serves as a DNA repair gene, and its function is altered by formation of BCR-ABL. This hypothesis was investigated using cell lines stably transfected with the BCR-ABL gene, and their parental counterparts (MBA-1 vs. M07E and Bcr-AblT1 vs. 4A2+pZAP), and several DNA repair assays: the Comet assay, a radioinimunoassay for UV-induced cyclobutane pyrimidine dimers (CPDs), and clonogenic assays. Comet assays demonstrated that, after exposure to either ultraviolet (UV)-C (0.5 to 10.0 joules m −2) or to gamma radiation (200–1000 rads) there was greater efficiency of DNA repair in the BCR-ABL-transfected cells compared to their parental controls. Furthermore, after UVC-irradiation, there was less production of CPDs, and a more rapid disappearance of these adducts in BCR-ABL-bearing cells. UV survival, as reflected by clonogenic assays, was also greater in the BCR-ABL-transfected cells. Taken together, these results indicate that, in our systems, BCR-ABL confers resistance to UVC-induced damage in cells, and increases DNA repair efficiency in response to both UVC- and gamma-irradiation. ^

In vitro repair of oxidative DNA damage by human nucleotide excision repair system: Possible explanation for neurodegeneration in Xeroderma pigmentosum patients

Xeroderma pigmentosum (XP) patients fail to remove pyrimidine dimers caused by sunlight and, as a consequence, develop multiple cancers in areas exposed to light. The second most common sign, present in 20–30% of XP patients, is a set of neurological abnormalities caused by neuronal death in the central and peripheral nervous systems. Neural tissue is shielded from sunlight-induced DNA damage, so the cause of neurodegeneration in XP patients remains unexplained. In this study, we show that two major oxidative DNA lesions, 8-oxoguanine and thymine glycol, are excised from DNA in vitro by the same enzyme system responsible for removing pyrimidine dimers and other bulky DNA adducts. Our results suggest that XP neurological disease may be caused by defective repair of lesions that are produced in nerve cells by reactive oxygen species generated as by-products of an active oxidative metabolism.

Developmental disorder associated with increased cellular nucleotidase activity

Four unrelated patients are described with a syndrome that included developmental delay, seizures, ataxia, recurrent infections, severe language deficit, and an unusual behavioral phenotype characterized by hyperactivity, short attention span, and poor social interaction. These manifestations appeared within the first few years of life. Each patient displayed abnormalities on EEG. No unusual metabolites were found in plasma or urine, and metabolic testing was normal except for persistent hypouricosuria. Investigation of purine and pyrimidine metabolism in cultured fibroblasts derived from these patients showed normal incorporation of purine bases into nucleotides but decreased incorporation of uridine. De novo synthesis of purines and cellular phosphoribosyl pyrophosphate content also were moderately decreased. The distribution of incorporated purines and pyrimidines did not reveal a pattern suggestive of a deficient enzyme activity. Assay of individual enzymes in fibroblast lysates showed no deficiencies. However, the activity of cytosolic 5′-nucleotidase was elevated 6- to 10-fold. Based on the possibility that the observed increased catabolic activity and decreased pyrimidine salvage might be causing a deficiency of pyrimidine nucleotides, the patients were treated with oral pyrimidine nucleoside or nucleotide compounds. All patients showed remarkable improvement in speech and behavior as well as decreased seizure activity and frequency of infections. A double-blind placebo trial was undertaken to ascertain the efficacy of this supplementation regimen. Upon replacement of the supplements with placebo, all patients showed rapid regression to their pretreatment states. These observations suggest that increased nucleotide catabolism is related to the symptoms of these patients, and that the effects of this increased catabolism are reversed by administration of uridine.

Photorepair mutants of Arabidopsis

UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6–4) pyrimidinone dimer (6–4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6–4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6–4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6–4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system.

The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA

tRNA pseudouridine synthase I (ΨSI) catalyzes the conversion of uridine to Ψ at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. ΨSI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNAPhe containing FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-tRNA. The covalent adduct between FUra-tRNA and ΨSI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluorouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for ΨS.

Human cells compromised for p53 function exhibit defective global and transcription-coupled nucleotide excision repair, whereas cells compromised for pRb function are defective only in global repair

After exposure to DNA-damaging agents, the p53 tumor suppressor protects against neoplastic transformation by inducing growth arrest and apoptosis. A series of investigations has also demonstrated that, in UV-exposed cells, p53 regulates the removal of DNA photoproducts from the genome overall (global nucleotide excision repair), but does not participate in an overlapping pathway that removes damage specifically from the transcribed strand of active genes (transcription-coupled nucleotide excision repair). Here, the highly sensitive ligation-mediated PCR was employed to quantify, at nucleotide resolution, the repair of UVB-induced cyclobutane pyrimidine dimers (CPDs) in genetically p53-deficient Li–Fraumeni skin fibroblasts, as well as in human lung fibroblasts expressing the human papillomavirus (HPV) E6 oncoprotein that functionally inactivates p53. Lung fibroblasts expressing the HPV E7 gene product, which similarly inactivates the retinoblastoma tumor-suppressor protein (pRb), were also investigated. pRb acts downstream of p53 to mediate G1 arrest, but has no demonstrated role in DNA repair. Relative to normal cells, HPV E6-expressing lung fibroblasts and Li–Fraumeni skin fibroblasts each manifested defective CPD repair along both the transcribed and nontranscribed strands of the p53 and/or c-jun loci. HPV E7-expressing lung fibroblasts also exhibited reduced CPD removal, but only along the nontranscribed strand. Our results provide striking evidence that transcription-coupled repair, in addition to global repair, are p53-dependent in UV-exposed human fibroblasts. Moreover, the observed DNA-repair defect in HPV E7-expressing cells reveals a function for this oncoprotein in HPV-mediated carcinogenesis, and may suggest a role for pRb in global nucleotide excision repair.

Ozone depletion and UVB radiation: Impact on plant DNA damage in southern South America

The primary motivation behind the considerable effort in studying stratospheric ozone depletion is the potential for biological consequences of increased solar UVB (280–315 nm) radiation. Yet, direct links between ozone depletion and biological impacts have been established only for organisms of Antarctic waters under the influence of the ozone “hole;” no direct evidence exists that ozone-related variations in UVB affect ecosystems of temperate latitudes. Indeed, calculations based on laboratory studies with plants suggest that the biological impact of ozone depletion (measured by the formation of cyclobutane pyrimidine dimers in DNA) is likely to be less marked than previously thought, because UVA quanta (315–400 nm) may also cause significant damage, and UVA is unaffected by ozone depletion. Herein, we show that the temperate ecosystems of southern South America have been subjected to increasingly high levels of ozone depletion during the last decade. We found that in the spring of 1997, despite frequent cloud cover, the passages of the ozone hole over Tierra del Fuego (55° S) caused concomitant increases in solar UV and that the enhanced ground-level UV led to significant increases in DNA damage in the native plant Gunnera magellanica. The fluctuations in solar UV explained a large proportion of the variation in DNA damage (up to 68%), particularly when the solar UV was weighted for biological effectiveness according to action spectra that assume a sharp decline in quantum efficiency with increasing wavelength from the UVB into the UVA regions of the spectrum.

Enzyme plus light therapy to repair DNA damage in ultraviolet-B-irradiated human skin

Ultraviolet-B (UVB) (290–320 nm) radiation-induced cyclobutane pyrimidine dimers within the DNA of epidermal cells are detrimental to human health by causing mutations and immunosuppressive effects that presumably contribute to photocarcinogenesis. Conventional photoprotection by sunscreens is exclusively prophylactic in nature and of no value once DNA damage has occurred. In this paper, we have therefore assessed whether it is possible to repair UVB radiation-induced DNA damage through topical application of the DNA-repair enzyme photolyase, derived from Anacystis nidulans, that specifically converts cyclobutane dimers into their original DNA structure after exposure to photoreactivating light. When a dose of UVB radiation sufficient to induce erythema was administered to the skin of healthy subjects, significant numbers of dimers were formed within epidermal cells. Topical application of photolyase-containing liposomes to UVB-irradiated skin and subsequent exposure to photoreactivating light decreased the number of UVB radiation-induced dimers by 40–45%. No reduction was observed if the liposomes were not filled with photolyase or if photoreactivating exposure preceded the application of filled liposomes. The UVB dose administered resulted in suppression of intercellular adhesion molecule-1 (ICAM-1), a molecule required for immunity and inflammatory events in the epidermis. In addition, in subjects hypersensitive to nickel sulfate, elicitation of the hypersensitivity reaction in irradiated skin areas was prevented. Photolyase-induced dimer repair completely prevented these UVB radiation-induced immunosuppressive effects as well as erythema and sunburn-cell formation. These studies demonstrate that topical application of photolyase is effective in dimer reversal and thereby leads to immunoprotection.

A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus

Members of the Src family of nonreceptor protein tyrosine kinases (PTKs) have been implicated in the regulation of cellular excitability and synaptic plasticity. We have investigated the role of these PTKs in in vitro models of epileptiform activity. Spontaneous epileptiform discharges were induced in vitro in the CA3 region of rat hippocampal slices by superfusion with the potassium channel blocker 4-aminopyridine in Mg2+-free medium. In hippocampal slices treated in this fashion, Src kinase activity was increased and the frequency of epileptiform discharges could be greatly reduced by inhibitor of the Src family of PTKs, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), but not by the inactive structural analog 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3). 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine also reduced epileptiform activity induced by either 4-aminopyridine or Mg2+-free medium alone. These observations demonstrate a role for Src family PTKs in the pathophysiology of epilepsy and suggest potential therapeutic targets for antiepileptic therapy.

UV-damage-mediated induction of homologous recombination in Arabidopsis is dependent on photosynthetically active radiation

Plants are continuously subjected to UV-B radiation (UV-B; 280–320 nm) as a component of sunlight causing damage to the genome. For elimination of DNA damage, a set of repair mechanisms, mainly photoreactivation, excision, and recombination repair, has evolved. Whereas photoreactivation and excision repair have been intensely studied during the last few years, recombination repair, its regulation, and its interrelationship with photoreactivation in response to UV-B-induced DNA damage is still poorly understood. In this study, we analyzed somatic homologous recombination in a transgenic Arabidopsis line carrying a β-glucuronidase gene as a recombination marker and in offsprings of crosses of this line with a photolyase deficient uvr2–1 mutant. UV-B radiation stimulated recombination frequencies in a dose-dependent manner correlating linearly with cyclobutane pyrimidine dimer (CPD) levels. Genetic deficiency for CPD-specific photoreactivation resulted in a drastic increase of recombination events, indicating that homologous recombination might be directly involved in eliminating CPD damage. UV-B irradiation stimulated recombination mainly in the presence of photosynthetic active radiation (400–700 nm) irrespective of photolyase activities. Our results suggest that UV-B-induced recombination processes may depend on energy supply derived from photosynthesis.

Efficient translesion replication in the absence of Escherichia coli Umu proteins and 3′–5′ exonuclease proofreading function

Translesion replication (TR) past a cyclobutane pyrimidine dimer in Escherichia coli normally requires the UmuD′2C complex, RecA protein, and DNA polymerase III holoenzyme (pol III). However, we find that efficient TR can occur in the absence of the Umu proteins if the 3′–5′ exonuclease proofreading activity of the pol III ɛ-subunit also is disabled. TR was measured in isogenic uvrA6 ΔumuDC strains carrying the dominant negative dnaQ allele, mutD5, or ΔdnaQ spq-2 mutations by transfecting them with single-stranded M13-based vectors containing a specifically located cis-syn T–T dimer. As expected, little TR was observed in the ΔumuDC dnaQ+ strain. Surprisingly, 26% TR occurred in UV-irradiated ΔumuDC mutD5 cells, one-half the frequency found in a uvrA6 umuDC+mutD5 strain. lexA3 (Ind−) derivatives of the strains showed that this TR was contingent on two inducible functions, one LexA-dependent, responsible for ≈70% of the TR, and another LexA-independent, responsible for the remaining ≈30%. Curiously, the ΔumuDC ΔdnaQ spq-2 strain exhibited only the LexA-independent level of TR. The cause of this result appears to be the spq-2 allele, a dnaE mutation required for viability in ΔdnaQ strains, since introduction of spq-2 into the ΔumuDC mutD5 strain also reduces the frequency of TR to the LexA-independent level. The molecular mechanism responsible for the LexA-independent TR is unknown but may be related to the UVM phenomenon [Palejwala, V. A., Wang, G. E., Murphy, H. S. & Humayun, M. Z. (1995) J. Bacteriol. 177, 6041–6048]. LexA-dependent TR does not result from the induction of pol II, since TR in the ΔumuDC mutD5 strain is unchanged by introduction of a ΔpolB mutation.

MethDB—a public database for DNA methylation data

Methylation of cytosine in the 5 position of the pyrimidine ring is a major modification of the DNA in most organisms. In eukaryotes, the distribution and number of 5-methylcytosines (5mC) along the DNA is heritable but can also change with the developmental state of the cell and as a response to modifications of the environment. While DNA methylation probably has a number of functions, scientific interest has recently focused on the gene silencing effect methylation can have in eukaryotic cells. In particular, the discovery of changes in the methylation level during cancer development has increased the interest in this field. In the past, a vast amount of data has been generated with different levels of resolution ranging from 5mC content of total DNA to the methylation status of single nucleotides. We present here a database for DNA methylation data that attempts to unify these results in a common resource. The database is accessible via WWW (http://www.methdb.de). It stores information about the origin of the investigated sample and the experimental procedure, and contains the DNA methylation data. Query masks allow for searching for 5mC content, species, tissue, gene, sex, phenotype, sequence ID and DNA type. The output lists all available information including the relative gene expression level. DNA methylation patterns and methylation profiles are shown both as a graphical representation and as G/A/T/C/5mC-sequences or tables with sequence positions and methylation levels, respectively.

Mutagenesis of the peptidyltransferase center of 23S rRNA: the invariant U2449 is dispensable

U2449 is one of many invariant residues in the central loop of domain V of 23S rRNA, a region that constitutes part of the peptidyltransferase center of the ribosome. In Escherichia coli, this U is post-transcriptionally modified to dihydrouridine (D) and is the only D modification found in E.coli rRNAs. To analyze the role of this base and its modification in ribosomal function, all three base substitutions were constructed on a plasmid copy of the rrnB operon and assayed for their ability to support cell growth in a strain of E.coli lacking chromosomal rrn operons. Both purine substitution mutations were not viable. However, growth and antibiotic sensitivity of cells expressing only the mutant D2449C rRNA was indistinguishable from wild type. We conclude that while a pyrimidine is required at position 2449 for proper ribosomal function, the D modification is dispensable.

A thermostable endonuclease III homolog from the archaeon Pyrobaculum aerophilum

Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100°C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80–160 mM NaCl and 70°C, PaNth displays DNA glycosylase/β-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.