Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice

We have established xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair (NER) deficiency, which rapidly developed skin tumors when exposed to a low dose of chronic UV like XP-A patients, confirming that the NER process plays an important role in preventing UVB-induced skin cancer. To examine the in vivo mutation in the UVB-irradiated epidermis, we established XPA (−/−), (+/−) and (+/+) mice carrying the Escherichia coli rpsL transgene with which the mutation frequencies and spectra in the UVB-irradiated epidermal tissue can be examined conveniently. The XPA (−/−) mice showed a higher frequency of UVB-induced mutation in the rpsL transgene with a low dose (150 J/m²) of UVB-irradiation than the XPA (+/−) and (+/+) mice, while, at a high dose (900 J/m²) they showed almost the same frequency of mutation as the XPA (+/−) and (+/+) mice, probably because of cell death in the epidermis of the XPA (−/−) mice. However, CC→TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the XPA (−/−) mice than the XPA (+/−) and (+/+) mice at both doses of UVB. This rpsL/XPA mouse system will be useful for further analyzing the role of NER in the mutagenesis and carcinogenesis induced by various carcinogens.     

Role of nucleotide excision repair deficiency in intestinal tumorigenesis in multiple intestinal neoplasia (Min) mice

Mice deficient in the Xeroderma pigmentosum group A (Xpa) gene are defective in nucleotide excision repair (NER) and highly susceptible to skin carcinogenesis after dermal exposure to UV light or chemicals. Min (multiple intestinal neoplasia) mice, heterozygous for a germline nonsense mutation in the tumor suppressor gene adenomatous polyposis coli (Apc), develop intestinal tumors spontaneously and show additional intestinal tumors after exposure to the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). In this study, we investigated the impact of loss of XPA function on PhIP-induced intestinal tumorigenesis in F1 offspring of Min/+ (Apc^+/−) mice crossed with Xpa gene-deficient mice. Apc^+/− mice lacking both alleles of Xpa had higher susceptibility towards toxicity of PhIP, higher levels of PhIP–DNA adducts in the middle and distal small intestines, as well as in liver, and a higher number of small intestinal tumors at 11 weeks, compared with Apc^+/− mice with one or two intact Xpa alleles. Localization of tumors was not affected, being highest in middle and distal small intestines in all genotypes. At 11 weeks of age, the number of spontaneous intestinal tumors was not significantly increased by homozygous loss of Xpa, but untreated Apc^+/−/Xpa^−/− mice had significantly shorter life-spans than their XPA-proficient littermates. Heterozygous loss of Xpa did not affect any of the measured end points. In conclusion, the Xpa gene and the NER pathway are involved in repair of bulky PhIP–DNA adducts in the intestines and the liver, and most probably of DNA lesions leading to spontaneous intestinal tumors. These results confirm a role of the NER pathway also in protection against cancer in internal organs, additional to its well-known importance in protection against skin cancer. An effect of Apc^+/− on adduct levels, additional to that of Xpa^−/−, indicates that the truncated APC protein may affect a repair pathway other than NER.     

Studies of in vivo mutations in rpsL transgene in UVB-irradiated epidermis of XPA-deficient mice

We have established xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair (NER) deficiency, which rapidly developed skin tumors when exposed to a low dose of chronic UV like XP-A patients, confirming that the NER process plays an important role in preventing UVB-induced skin cancer. To examine the in vivo mutation in the UVB-irradiated epidermis, we established XPA (−/−), (+/−) and (+/+) mice carrying the Escherichia coli rpsL transgene with which the mutation frequencies and spectra in the UVB-irradiated epidermal tissue can be examined conveniently. The XPA (−/−) mice showed a higher frequency of UVB-induced mutation in the rpsL transgene with a low dose (150 J/m²) of UVB-irradiation than the XPA (+/−) and (+/+) mice, while, at a high dose (900 J/m²) they showed almost the same frequency of mutation as the XPA (+/−) and (+/+) mice, probably because of cell death in the epidermis of the XPA (−/−) mice. However, CC→TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the XPA (−/−) mice than the XPA (+/−) and (+/+) mice at both doses of UVB. This rpsL/XPA mouse system will be useful for further analyzing the role of NER in the mutagenesis and carcinogenesis induced by various carcinogens.     

Mutagenesis and carcinogenesis in nucleotide excision repair-deficient XPA knock out mice

Mice with a defect in the xeroderma pigmentosum group A (XPA) gene have a complete deficiency in nucleotide excision repair (NER). As such, these mice mimic the human XP phenotype in that they have a >1000-fold higher risk of developing UV-induced skin cancer. Besides being UV-sensitive, XPA^−/− mice also develop internal tumors when they are exposed to chemical carcinogens. To investigate the effect of a total NER deficiency on the induction of gene mutations and tumor development, we crossed XPA^−/− mice with transgenic lacZ/pUR288 mutation-indicator mice. The mice were treated with various agents and chemicals like UV-B, benzo[a]pyrene and 2-aceto-amino-fluorene. Gene mutation induction in several tumor target- and non-target tissues was determined in both the bacterial lacZ reporter gene and in the endogenous Hprt gene. Furthermore, alterations in the p53- and ras genes were determined in UV-induced skin tumors of XPA^−/− mice. In this work, we review these results and discuss the applicability and reliability of enhanced gene mutant frequencies as early indicators of tumorigenesis.     

Additive roles of XPA and MSH2 genes in UVB-induced skin tumorigenesis in mice

We have made xeroderma pigmentosum group A gene (XPA)-knockout mice (XPA(-/-) mice). The XPA(-/-) mice had no detectable activity for nucleotide excision repair (NER) and showed a high incidence of UVB-induced skin tumorigenesis. We have also found that cell lines derived from skin cancers in UVB-irradiated XPA(-/-) mice become tolerant to UV-irradiation and showed abnormal UV-induced cell cycle checkpoints and decreased mismatch repair (MMR) activity. These results suggested that the MMR-downregulation may help cells escape killing by UV-irradiation and thus MMR-deficient clones are selected for during the tumorigenic transformation of XPA(-/-) cells. In this report, we examined whether the incidence of UVB-induced skin tumorigenesis is enhanced in XPA(-/-)MSH2(-/-), XPA(-/-) and MSH2(-/-) mice when compared with that in wild-type mice. Our results indicate that the MSH2-deficiency caused a high incidence of spontaneous and UVB-induced skin tumorigenesis and the XPA and MSH2 genes have additive roles in the UV-induced skin tumorigenesis.     

α-N-Methylation of Damaged DNA-binding Protein 2 (DDB2) and Its Function in Nucleotide Excision Repair

DDB2 exhibits a high affinity toward UV-damaged DNA, and it is involved in the initial steps of global genome nucleotide excision repair. Mutations in the DDB2 gene cause the genetic complementation group E of xeroderma pigmentosum, an autosomal recessive disease manifested clinically by hypersensitivity to sunlight exposure and an increased predisposition to skin cancer. Here we found that, in human cells, the initiating methionine residue in DDB2 was removed and that the N-terminal alanine could be methylated on its α-amino group in human cells, with trimethylation being the major form. We also demonstrated that the α-N-methylation of DDB2 is catalyzed by the N-terminal RCC1 methyltransferase. In addition, a methylation-defective mutant of DDB2 displayed diminished nuclear localization and was recruited at a reduced efficiency to UV-induced cyclobutane pyrimidine dimer foci. Moreover, loss of this methylation conferred compromised ATM (ataxia telangiectasia mutated) activation, decreased efficiency in cyclobutane pyrimidine dimer repair, and elevated sensitivity of cells toward UV light exposure. Our study provides new knowledge about the posttranslational regulation of DDB2 and expands the biological functions of protein α-N-methylation to DNA repair.     

High levels of oxidatively generated DNA damage 8,5′-cyclo-2′-deoxyadenosine accumulate in the brain tissues of xeroderma pigmentosum group A gene-knockout mice

Xeroderma pigmentosum (XP) is a genetic disorder associated with defects in nucleotide excision repair, a pathway that eliminates a wide variety of helix-distorting DNA lesions, including ultraviolet-induced pyrimidine dimers. In addition to skin diseases in sun-exposed areas, approximately 25% of XP patients develop progressive neurological disease, which has been hypothesized to be associated with the accumulation of an oxidatively generated type of DNA damage called purine 8,5′-cyclo-2′-deoxynucleoside (cyclopurine). However, that hypothesis has not been verified. In this study, we tested that hypothesis by using the XP group A gene-knockout (Xpa^−/−) mouse model. To quantify cyclopurine lesions in this model, we previously established an enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody (CdA-1) that specifically recognizes 8,5′-cyclo-2′-deoxyadenosine (cyclo-dA). By optimizing conditions, we increased the ELISA sensitivity to a detection limit of ˜one cyclo-dA lesion/10⁶ nucleosides. The improved ELISA revealed that cyclo-dA lesions accumulate with age in the brain tissues of Xpa^−/− and of wild-type (wt) mice, but there were significantly more cyclo-dA lesions in Xpa^−/− mice than in wt mice at 6, 24 and 29 months of age. These findings are consistent with the long-standing hypothesis that the age-dependent accumulation of endogenous cyclopurine lesions in the brain may be critical for XP neurological abnormalities.     

Inhibition of protein synthesis does not antagonize induction of UV-induced sister-chromatid exchange in xeroderma pigmentosum cells

Cycloheximide strongly antagonizes the induction of sister-chromatid exchanges by ethyl methanesulfonate or mitomycin C in human skin fibroblast and xeroderma pigmentosum cells (group A). Analogous behavior has been observed in several other species including Chinese hamster and plant cells. This report documents an exception to that pattern: cycloheximide fails to antagonize UV-induced sister chromatid exchange in xeroderma pigmentosum cells, whereas it does in normal human skin fibroblast cells. A genetic defect in these cells is postulated to alter the UV-mediated DNA recombination process.     

Detection and repair of a UV-induced photosensitive lesion in the DNA of human cells

Irradiation with UV light results in damage to the DNA of human cells. The most numerous lesions are pyrimidine dimers; however, other lesions are known to occur and may contribute to the overall deleterious effect of UV irradiation. We have observed evidence of a UV-induced lesion other than pyrimidine dimers in the DNA of human cells by measuring DNA strand breaks induced by irradiating with 313-nm light following UV (254-nm) irradiation. These breaks, measured by alkaline sucrose sedimentation, increased linearly with the dose of UV light over the range tested (10-40 J/m2). The breaks cannot be photolytically induced 5 h after a UV dose of 20 J/m2 in normal cells; however, in xeroderma pigmentosum variant cells, the breaks are inducible for up to 24 h after UV irradiation. Xeroderma pigmentosum group A cells in the same 5-h period show an increase in the number of strand breaks seen with 313-nm light photolysis from about 2 to 4 breaks/10(9) dalton DNA. These breaks can then be induced for up to 24 h. These data suggest that, in normal cells, the lesion responsible for this effect is rapidly repaired or altered; whereas, in xeroderma pigmentosum variant cells it seems to remain unchanged. Some change apparently occurs in the DNA of xeroderma pigmentosum group A cells which results in an increase in photolability. These data indicate a deficiency in DNA repair of xeroderma pigmentosum variant cells as well as in xeroderma pigmentosum group A cells.     

Sunless skin tanning with dihydroxyacetone delays broad-spectrum ultraviolet photocarcinogenesis in hairless mice

Sunless tanning with dihydroxyacetone (DHA) is not considered to be a sunscreen although it does absorb parts of the ultraviolet (UV) spectrum. We investigated the protection with topical application of DHA against solar UV-induced skin carcinogenesis in lightly pigmented hairless hr/hr C3H/Tif mice. Broad-spectrum UV radiation, simulating the UV part of the solar spectrum was obtained from one Philips TL12 and five Bellarium-S SA-1-12 tubes. Three groups of mice were UV-exposed four times a week to a dose-equivalent of four times the standard erythema dose (SED), without or with application of 5 or 20% DHA only twice a week. Similarly, three groups of mice were treated with DHA and irradiated with a high UV dose (8 SED), simulating a skin burn. Two groups (controls) were not irradiated, but either left untreated or treated with 20% DHA alone. The UV-induced skin pigmentation by melanogenesis could easily be distinguished from DHA-induced browning and was measured by a non-invasive, semi-quantitative method. Application of 20% DHA reduced by 63% the pigmentation produced by 4 SED, however, only by 28% the pigmentation produced by 8 SED. Furthermore, topical application of 20% DHA significantly delayed the time to appearance of the first tumor ≥1 mm (P=0.0012) and the time to appearance of the third tumor (P=2×10⁻⁶) in mice irradiated with 4 SED. However, 20% DHA did not delay tumor development in mice irradiated with 8 SED. Application of 5% DHA did not influence pigmentation or photocarcinogenesis.In conclusion, this is the first study to show that the superficial skin coloring generated by frequent topical application of DHA in high concentrations may delay skin cancer development in hairless mice irradiated with moderate UV doses.     

Unique DNA repair properties of a xeroderma pigmentosum revertant.

A group A xeroderma pigmentosum revertant with normal sensitivity was created by chemical mutagenesis. It repaired (6-4) photoproducts normally but not pyrimidine dimers and had near normal levels of repair replication, sister chromatid exchange, and mutagenesis from UV light. The rate of UV-induced mutation in a shuttle vector, however, was as high as the rate in the parental xeroderma pigmentosum cell line.     

DNA damage initiates photobiologic reactions in the skin.

Ultraviolet (UV) radiation can induce acute and chronic photobiologic reactions in the absence of exogenous chromophores. Nuclear DNA is a major chromophore to initiate UV-induced physiologic reactions. The XPA-gene deficient mouse, an animal model of xeroderma pigmentosum, develops increased photobiologic reactions including acute inflammation, immunosuppression and skin cancers, because of the defect in the excision repair of UV-induced DNA lesions.     

Effects of neuropathy on high-voltage-activated Ca current in sensory neurones

Voltage-dependent Ca²⁺ channels (VDCCs) have emerged as targets to treat neuropathic pain; however, amongst VDCCs, the precise role of the Ca_V2.3 subtype in nociception remains unproven. Here, we investigate the effects of partial sciatic nerve ligation (PSNL) on Ca²⁺ currents in small/medium diameter dorsal root ganglia (DRG) neurones isolated from Ca_V2.3(−/−) knock-out and wild-type (WT) mice. DRG neurones from Ca_V2.3(−/−) mice had significantly reduced sensitivity to SNX-482 versus WT mice. DRGs from Ca_V2.3(−/−) mice also had increased sensitivity to the Ca_V2.2 VDCC blocker ω-conotoxin. In WT mice, PSNL caused a significant increase in ω-conotoxin-sensitivity and a reduction in SNX-482-sensitivity. In Ca_V2.3(−/−) mice, PSNL caused a significant reduction in ω-conotoxin-sensitivity and an increase in nifedipine sensitivity. PSNL-induced changes in Ca²⁺ current were not accompanied by effects on voltage-dependence of activation in either Ca_V2.3(−/−) or WT mice. These data suggest that Ca_V2.3 subunits contribute, but do not fully underlie, drug-resistant (R-type) Ca²⁺ current in these cells. In WT mice, PSNL caused adaptive changes in Ca_V2.2- and Ca_V2.3-mediated Ca²⁺ currents, supporting roles for these VDCCs in nociception during neuropathy. In Ca_V2.3(−/−) mice, PSNL-induced changes in Ca_V1 and Ca_V2.2 Ca²⁺ current, consistent with alternative adaptive mechanisms occurring in the absence of Ca_V2.3 subunits.     

The effects of microsomal prostaglandin E synthase-1 deletion in acute cardiac ischemia in mice

The goal of the present study was to assess how genetic loss of microsomal prostaglandin E₂ synthase-1 (mPGES-1) affects acute cardiac ischemic damage after coronary occlusion in mice. Wild type (WT), heterozygous (mPGES-1^+/−), and homozygous (mPGES-1^−/−) knockout mice were subjected to left coronary artery occlusion. At 24 h, myocardial infarct (MI) volume was measured histologically. Post-MI survival, plasma levels of creatine phosphokinase (CPK) and cardiac troponin-I, together with MI size, were similar in WT, mPGES-1^+/− and mPGES-1^−/− mice. In contrast, post-MI survival was reduced in mPGES-1^−/− mice pretreated with I prostanoid receptor (IP) antagonist (12/16) compared with vehicle-treated controls (13/13 mPGES-1^−/−) together with increased CPK and cardiac troponin-I release. The deletion of mPGES-1 in mice results in increased prostacyclin I₂ (PGI₂) formation and marginal effects on the circulatory prostaglandin E₂ (PGE₂) level. We conclude that loss of mPGES-1 results in increased PGI₂ formation, and in contrast to inhibition of PGI₂, without worsening acute cardiac ischemic injury.     

Synergistic interactions between XPC and p53 mutations in double-mutant mice: neural tube abnormalities and accelerated UV radiation-induced skin cancer

The significance of DNA repair to human health has been well documented by studies on xeroderma pigmentosum (XP) patients, who suffer a dramatically increased risk of cancer in sun-exposed areas of their skin [1] and [2]. This autosomal recessive disorder has been directly associated with a defect in nucleotide excision–repair (NER) [1] and [2]. Like human XP individuals, mice carrying homozygous mutations in XP genes manifest a predisposition to skin carcinogenesis following exposure to ultraviolet (UV) radiation [3], [4] and [5]. Recent studies have suggested that, in addition to roles in apoptosis [6] and cell-cycle checkpoint control [7] in response to DNA damage, p53 protein may modulate NER [8]. Mutations in the p53 gene have been observed in 50% of all human tumors [9] and have been implicated in both the early [10] and late [11] stages of skin cancer. To examine the consequences of a combined deficiency of the XPC and the p53 proteins in mice, we generated double-mutant animals. We document a spectrum of neural tube defects in XPC p53 mutant embryos. Additionally, we show that, following exposure to UV-B radiation, XPC p53 mutant mice have more severe solar keratosis and suffer accelerated skin cancer compared with XPC mutant mice that are wild-type with respect to p53.     

Ultraviolet light-induced chromosomal aberrations in cultured cells from Cockayne syndrome and complementation group C xeroderma pigmentosum patients: lack of correlation with cancer susceptibility.

Both Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are inherited diseases with defective repair of damage induced in DNA by UV. Patients with XP, but not those with CS, have an increased susceptibility to formation of sunlight-induced skin tumors. We determined the frequency of UV-induced chromosomal aberrations in cultured lymphoblastoid cell lines from five CS patients and three complementation-group-C XP patients to determine whether such aberrations were abnormally increased only in the XP cells. We found that CS cells had the same abnormally increased number of induced aberrations as the XP cells, indicating that the number of UV-induced aberrations in XP group C cells does not account for the susceptibility of these XP patients to sunlight-induced skin cancer.     

Genetic recombination of herpes simplex virus, the role of the host cell and UV-irradiation of the virus

Summary Recombination frequencies for two sets of genetic markers of herpes simplex virus were determined in various host cells with and without ultraviolet irradiation of the virus. UV irradiation increased the recombination frequency in all the cell types studied in direct proportion to the unrepaired lethal damage. In human skin fibroblasts derived from a patient with xeroderma pigmentosum (XP) of complementation group A, a given dose of UV stimulated recombination more than that in fibroblasts from normal individuals. On the other hand, UV stimulation of HSV recombination was slightly less than normal in fibroblasts derived from a patient with a variant form XP and from an ataxia telangiectasia patient. Caffeine, an agent known to inhibit repair of UV damage, reduced recombination in most of the cell types studied but did not suppress the UV-induced increase in recombination. These findings suggest that for virus DNA with the same number of unrepaired UV-lesions, each of the tested cell types promoted HSV-recombination to an equivalent extent.     

Performance and kinetic evaluation of anaerobic moving bed biofilm reactor for treating milk permeate from dairy industry

High strength milk permeate derived from ultra-filtration based cheese making process was treated in an anaerobic moving bed biofilm reactor (AMBBR) under mesophilic (35 °C) condition. Total chemical oxygen demand (TCOD) removal efficiencies of 86.3–73.2% were achieved at organic loading rates (OLR) of 2.0–20.0 g TCOD L⁻¹ d⁻¹. A mass balance model gave values of methane yield coefficient (Y_G/S) and cell maintenance coefficient (k_m) of 0.341 L CH₄ g⁻¹ TCOD_removed and 0.1808 g TCOD_removed g⁻¹ VSS d⁻¹, respectively. The maximum substrate utilization rate U_max was determined as 89.3 g TCOD L⁻¹ d⁻¹ by a modified Stover–Kincannon model. Volumetric methane production rates (VMPR) were shown to correlate with the biodegradable TCOD concentration through a Michaelis–Menten type equation. Moreover, based on VMPR and OLR removed from the reactor, the sludge production yield was determined as 0.0794 g VSS g⁻¹ TCOD_removed.     

Unusual intramolecular N→O acyl group migration occurring during conjugation of (−)-DHMEQ with cysteine

Previously we found that (−)-DHMEQ, a specific NF-κB inhibitor, covalently bound to a specific cysteine of NF-κB component proteins. In the course of formation of the (−)-DHMEQ and protected cysteine conjugate, we observed an unusual intramolecular N→O acyl group migration.