A truncated human xeroderma pigmentosum complementation group A protein expressed from an adenovirus sensitizes human tumor cells to ultraviolet light and cisplatin.

Individuals with the genetic disease xeroderma pigmentosum (XP) have impaired nucleotide excision repair (NER). Group A XP cells are defective in the XPA protein essential for NER and serve, together with other NER proteins, as a nucleation factor for the demarcation of bulky DNA damage. Because XPA cells are extremely sensitive to UV and drugs that cause bulky DNA damage, the XPA protein is an attractive target for manipulating cellular sensitivity to certain cancer therapeutics, a concept that perhaps can be applied toward developing more effective cancer treatments. We have made a replication-defective adenovirus, AdCMV-FlagXPA(59-114), that expresses a truncated form of XPA encompassing amino acids 59-114 sufficient for binding to the excision repair cross-complementing protein 1 (ERCC1)/xeroderma pigmentosum complementation group F (XPF) nuclease essential for making an incision 5' of the damage. On the basis of previous work, it was expected that this truncated XPA protein would work as a decoy and impair NER and, thus, sensitize cells to UV and drugs that produce bulky DNA lesions. Because the truncated XPA protein is "tagged" with the Flag epitope, an anti-Flag antibody can be used to detect protein expression and to isolate proteins associated with the XPA complex. We show that relatively large quantities of truncated XPA protein are present in infected human lung carcinoma A549 cells 2-4 days postinfection. Moreover, in a pull-down assay using anti-Flag antibody, we show that ERCC1 is present in the FlagXPA complex but not in a complex isolated from cells infected with a control virus. Most importantly, cells infected with AdCMV-FlagXPA(59-114) are significantly more sensitive than control cells to UV-induced damage as determined by host-cell reactivation of UV-irradiated AdLacZ adenovirus and in a cytotoxicity assay that appears to be the result of aberrant processing of 6-4 photoproducts. Infected cells were also more sensitive to treatment with cisplatin, an important cancer drug. These results suggest that NER, and the XPA protein in particular, can be a direct target for sensitizing tumor cells to UV and cisplatin and perhaps also certain other clinically important drugs.

Alpha-tocopherol modulates lipoprotein cytotoxicity in obstructive nephropathy.

Oxidative stress in unilateral ureteral obstruction (UUO) contributes to the development of glomerular and tubulointerstitial lesions. The present study investigated whether oxidized low-density lipoprotein (oLDL) contributes to the pathogenesis of kidney injury in UUO, and whether alpha-tocopherol modulates such cytotoxicity and promotes repair. Male Sprague-Dawley rats weighing 100-125 g were assigned to three groups of 6 animals each: (1) sham, regular chow; (2) UUO, regular chow; and (3) UUO, alpha-tocopherol supplementation. We found a significant increase in the level of oxidative stress in the UUO group as measured by malondialdehyde (MDA) content in both plasma and kidneys. The LDL isolated from this group was cytotoxic to rat mesangial cells. The level of oxidation and cytotoxicity was significantly reduced when animals were treated with alpha-tocopherol. Plasma cholesterol concentration, kidney MDA, and transforming growth factor beta1 mRNA expression were all significantly increased in the UUO animals, and partially reduced in alpha-tocopherol-treated animals. Our data suggest that oxidative modification of LDL is associated with the renal injury in UUO. Taken together, our data support the concept that alpha-tocopherol can modulate LDL oxidation and its cytotoxic effects on rat mesangial cells in vitro.

Gene expression after intrarenal injection of plasmid DNA in the rat.

Effective gene therapy requires efficient delivery and expression of the necessary genetic information to the target tissue. We demonstrate here that plasmid DNA, injected as naked, uncomplexed DNA into the cortical region of rat kidney, or intravenously, is localized and expressed in the kidney. The plasmid pRSVZ contained the Rous sarcoma virus promoter and a reporter gene, the beta-galactosidase gene, derived from bacteria. The beta-galactosidase gene hydrolyzes the artificial substrate X-gal to produce an intense blue color in cells that have taken up and expressed the plasmid genes. We have used X-gal staining and Western blotting to study plasmid gene expression 1, 4, and 8 days and 6 months after intrarenal injection of 50 microg of plasmid DNA and at 1 and 4 days after intravenous injection. Expression was apparent in the kidneys and several other tissues 24 h after injection and persisted for at least 8 days; expressed proteins could still be detected in the injected kidney 6 months later. These observations were corroborated by use of a plasmid, pEGFP-Puro, harboring the cytomegalovirus promoter in conjunction with a different reporter gene, the green fluorescent protein (GFP). Histological localization and Western blotting analysis of GFP expression after intrarenal injection of pEGFP-Puro paralleled results obtained with the plasmid pRSVZ. Our findings support the suggestion that intrarenal or intravenous injection of naked plasmid DNA may be an effective means of delivering therapeutic genes to the kidney and several other tissues.

Diphenyleneiodium chloride blocks inflammatory cytokine-induced up-regulation of group IIA phospholipase A(2) in rat mesangial cells.

Inflammatory cytokines, interleukin 1beta and tumor necrosis factor-alpha, potently stimulate rat mesangial cells to express and secrete group IIA phospholipase A(2) (PLA(2)). Cytokine-induced up-regulation of PLA(2) has been blocked by inhibitors (antioxidants) of the transcription factor, nuclear factor-kappaB (NF-kappaB), suggesting a role for NF-kappaB in the regulation of group IIA PLA(2) expression. Reactive oxygen species such as H(2)O(2), which are elevated in mesangial cells after cytokine activation, can mimic cytokine-induced NF-kappaB activation. However, the source of reactive oxygen species generation in mesangial cells, produced by cytokine stimulation, has yet to be clarified. Recently, tumor necrosis factor-alpha has been demonstrated to increase superoxide radical generation in mesangial cells. Therefore, we hypothesized that a selective NADPH oxidase inhibitor, diphenyleneiodium chloride (DPI), could block cytokine-induced group IIA PLA(2) up-regulation by attenuating NF-kappaB binding. To test this hypothesis, we isolated rat mesangial cells and characterized them by ultrastructural and immunochemical methods. This homogeneous mesangial cell population was responsive to cytokine as evidenced by an increase in steady-state levels of group IIA PLA(2) mRNA and extracellular enzymatic activity over time. DPI (0.02-20 microM), added 90 min before cytokine activation, inhibited both group IIA PLA(2) mRNA and enzymatic activity in a concentration-dependent manner. By electrophoretic mobility shift analysis, cytokine activation also increased specific NF-kappaB binding to one of two NF-kappaB consensus elements in the rat group IIA PLA(2) promoter and also was suppressed by DPI pretreatment. Antibodies to NF-kappaB p65 (Rel A) and p50 (but not normal rabbit IgG) supershifted this retardation signal and verified the type of NF-kappaB species as the classical p50/p65 heterodimer.

Vitamin E suppresses oxidative stress and glomerulosclerosis in rat remnant kidney.

Previous studies have shown that reduction of renal mass in the rat remnant kidney model induces overproduction of transforming growth factor beta1 (TGFbeta1). We investigated whether an antioxidant, vitamin E, administered before the renal mass reduction, could prevent oxidative stress, reduce the overproduction of TGFbeta1, and mitigate against the subsequent glomerulosclerosis. Our results revealed that the oxidative stress, as measured by the change in plasma malondialdehyde, is significantly reduced by prior vitamin E dietary supplementation. Finally, our data show that dietary vitamin E supplementation ameliorates the rise in TGFbeta1 secondary to renal mass reduction and inhibits the glomerular sclerosis of the remnant kidney over the time course of this experiment.

Influence of alpha-tocopherol over the time course of experimental IgA nephropathy.

The present study investigated the pathogenesis and the time course of kidney injury in experimental IgA nephropathy. In order to determine an appropriate period in the course of experimental IgA nephropathy to study renal injury and repair, we examined proteinuria and IgA deposition in the renal mesangium after 4, 8, and 16 weeks of mucosal challenge by bovine gamma globulins (BGG) provided in the drinking water. The hallmark of IgA deposition in the mesangium was present after 4 weeks and 8 weeks of BGG inoculation, but by 16 weeks, the mesangial IgA deposition had resolved. In addition, we confirmed our previous report on the beneficial effects of alpha-tocopherol in reducing proteinuria in IgA nephropathy at 8 weeks, and extended this observation to investigate the effects of dietary supplementation of alpha-tocopherol at both 4 weeks and 16 weeks. Proteinuria resolved spontaneously at 16 weeks. There is oxidative stress, as suggested by the elevation in plasma and renal malondialdehyde content, and increased fibrogenic cytokine message, as suggested by elevated transforming growth factor beta1 mRNA. These increases were clearly blunted by alpha-tocopherol at both 4 weeks and 8 weeks. Treatment with alpha-tocopherol was associated with a significant reduction in the severity of proteinuria. Thus, our data suggest that the period between 4 and 8 weeks of BGG vaccination could be relevant in designing an appropriate model to study the molecular biology of the pathogenesis of renal injury and the effects of treatment. The 16-week model may be useful in exploring gene expression involved with spontaneous resolution.

Glomerulosclerosis in the remnant kidney rat is modulated by dietary alpha-tocopherol.

Glomerulosclerosis and tubulointerstitial injury are characteristic features seen in the subtotal (5/6) nephrectomy remnant kidney model in the rat. Oxidative stress from renal mass reduction contributes to the glomerular and tubular injury. Previous studies have clearly demonstrated the prevention or inhibition of such injury by an antioxidant such as alpha-tocopherol. However, few data are available on the ability of alpha-tocopherol to modulate or arrest progression of the established disease. This study examines whether alpha-tocopherol modulates glomerulosclerosis and tubulointerstitial injury when it is given 2 wk after renal damage has been established. The findings indicate that alpha-tocopherol has the capacity to modulate both tubulointerstitial injury and glomerulosclerosis, lower the elevated expression of transforming growth factor-beta1, and reduce plasma and kidney malondialdehyde concentration, the end product of lipid peroxidation. The results support the potential utility of alpha-tocopherol in reversing established glomerulosclerosis and tubulointerstitial injury in a remnant kidney model.

Effects of fish oil and alpha-tocopherol in immunoglobulin A nephropathy in the rat.

Alpha-tocopherol and fish oil have been reported to modulate the progression of IgA nephropathy in animals and humans. Because fish oil has been reported to exacerbate renal disease in subtotal nephrectomized rats, we investigated the effects of fish oil, with and without alpha-tocopherol, on the course of IgA nephropathy. Experimental IgA nephropathy was induced in male Sprague-Dawley rats, weighing 170-200 g, by oral and i.v. immunization with bovine gamma-globulin for 8 wk. IgA nephropathy was evidenced by hematuria, proteinuria, and IgA deposition in the mesangium. Standard rodent chow, containing 30 IU of alpha-tocopherol/kg of diet, was given to the control and IgA nephropathy rats. Fish oil (20% wt/wt), stripped of alpha-tocopherol preservative, was given to control and a second group of IgA nephropathy rats. Alternatively, corn oil or fish oil was supplemented with alpha-tocopherol at 100 IU/kg of diet and given to the third and fourth groups of IgA nephropathy rats. All animals were killed at 8 wk. Urinary protein excretion, plasma and kidney alpha-tocopherol concentrations, as well as glomerular planar area, and kidney transforming growth factor-beta1 mRNA were analyzed. As determined by reductions in proteinuria, glomerular planar area, and TGF-beta1 mRNA, fish oil with alpha-tocopherol ameliorated the renal injury induced by bovine gamma-globulin, whereas fish oil without alpha-tocopherol did not. Our findings support the importance of alpha-tocopherol, more so than fish oil, in mitigating the injury and promoting repair in experimental IgA nephropathy.

Neonatal edema.

Edema develops in the neonate from diverse clinical conditions; sometimes it heralds serious underlying disorders. In this review, we discuss the diagnosis and treatment of edema in the neonate.

Edema in childhood.

There are two types of edema: localized edema and generalized edema. The causes of generalized edema in childhood are diverse. Formation of generalized edema involves retention of sodium and water in the kidney. The treatment of generalized edema depends on the primary etiology. Supportive nutritional and medical therapies are needed to prevent further edema. These and related features of edema in childhood are discussed in this review.

Inhibition of transforming growth factor beta 1 induction by dietary vitamin E in unilateral ureteral obstruction in rats.

Free radical species associated with bilateral ureteral obstruction (BUO) are considered important in the pathogenesis of the glomerular and tubulointerstitial injury in BUO rats. We seek to test the hypothesis that the use of an easily administered antioxidant, vitamin E, at sufficient plasma concentrations, can decrease this release of free oxygen radicals in kidney tissue and ameliorate the increase of the fibrogenic cytokine, transforming growth factor beta-1 (TGF beta-1). We used the unilateral ureteral obstruction (UUO) rat model, because the presence of the uninjured contralateral kidney provides a nonuremic internal milieu, in contrast to the uremic, acidotic, and hypercholesterolemic BUO model. Compared to sham controls, the UUO animals showed a dramatic increase in renal cortical TGF beta-1 mRNA, as quantitated by Northern blot analysis with cyclophilin internal standards. This increase in TGF beta-1 mRNA was reversed in UUO rats treated with vitamin E. The plasma malondialdehyde (MDA) concentration, an index of lipid peroxidation and an indirect index of free radical release, was significantly elevated in UUO animals compared to sham animals. The vitamin E-treated UUO animals showed a significant decrease in both plasma and renal cortical tissue MDA content. Taken together, these findings provide evidence of the important biological role of reactive free radical species in the tubulointerstitial injury of UUO and the novel role of vitamin E in modulating the mRNA of the fibrogenic TGF beta-1 in obstructive uropathy.

Ionizing radiation activates nuclear factor kappa B but fails to produce an increase in human immunodeficiency virus gene expression in stably transfected human cells.

We have investigated the differential effects of ultraviolet light(UV) and ionizing radiation (IR) on human immunodeficiency virus type 1 (HIV) and c-jun expression in HIVcat/HeLa cells. This cell line harbors integrated copies of the chloramphenicol acetyltransferase (cat) gene under control of the HIV promoter. Both UV and IR increased the binding of nuclear proteins to an oligonucleotide spanning the HIV enhancer region nuclear factor kappa B sites, but only UV increased HIVcat steady-state mRNA and CAT activity. By comparison, transcription of the cellular c-jun gene increased after both types of radiation, but UV was at least 5-fold more effective than IR despite the fact that protein binding to an activator protein 1 oligonucleotide increased similarly after both UV and IR. The lack of HIVcat transcriptional response after IR does not appear to be the result of the repressor binding to upstream promoter elements since cells stably transfected with different HIV promoter deletions showed a lack of response to IR distinguishable from that of the intact promoter. While our findings indicate no correlation between increased binding of transcription factors to upstream promoter elements and increased expression of these genes after radiation, we did observe major differences in how UV and IR affected chromatin structure. UV produced extensive global chromatin decondensation, whereas IR did not, as seen in the microscope and determined by the increased susceptibility of chromatin to micrococcal nuclease digestion.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of two solitary long terminal repeats of murine leukemia virus type that are conserved in the chromosome of laboratory inbred mouse strains.

Twenty molecular clones containing sequences homologous to the long terminal repeats (LTRs) of the endogenous ecotropic murine leukemia virus (MuLV) of the RFM/Un mouse were isolated from a library of RFM/Un mouse spleen DNA in phage lambda. Three of these LTRs were not associated with any viral structural genes. Nucleotide sequence analysis demonstrated that they were solitary LTRs which were flanked by 4-bp directly repeated cellular sequences and which lacked primer binding sites. Two of the three subclones were found to be identical except for their orientations in the vector pBR322. Unique-sequence regions on either side of the two nonidentical elements were used to characterize their integration sites in genomic DNA. The solitary LTRs and their flanking regions were found to be conserved in a number of inbred mouse strains, including three strains known not to harbor endogenous ecotropic MuLV-type proviruses. Comparison of cleavage by the methylation-sensitive restriction enzyme SmaI and methylation-insensitive KpnI at the characteristic LTR SmaI/KpnI site suggested that at least one of these solitary LTRs is methylated to a lesser extent than are most endogenous proviral LTRs. These particular solitary LTRs, like endogenous proviral sequences, appear to be stably transmitted genetic elements.

An in vitro complementation assay for the Escherichia coli uvrD gene product.

An in vitro assay specific for the product of the uvrD gene of Escherichia coli has been developed. This assay, derived from properties of uvrD mutants revealed by in vivo experiments, is based on the necessity for a functional UvrD protein for complete rejoining of covalently closed circular DNA during the excision repair of UV-induced damage. Extracts prepared from gently lysed uvrD101 mutant cells are capable of restoring UV-damaged DNA to its covalently closed circular form when provided with a functional UvrD protein from other repair-deficient cell extracts or from partially purified protein fractions. This assay was employed to monitor the activity of the UvrD protein after several steps of fractionation. The partially purified UvrD protein does not complement extracts deficient in DNA polymerase I or temperature-sensitive in DNA ligase; it does, however, complement extracts from strains mutant at the uvrE and recL loci, which are considered allelic with the uvrD locus.

The effect of mutations in the uvrD cistron of Escherichia coli on repair resynthesis.

The resynthesis step of the excision repair pathway has been examined in Escherichia coli K12 strains isogenic except for mutations in the uvrD cistron. Strains mutant at the uvrD3, uvrD101, uvrE156, and recL152 loci exhibited slight but distinct differences in their response to ultraviolet radiation. The repair capacity of the uvrD101 mutant was given special attention. Repair resynthesis in this mutant was saturated at fluences greater than about 20 J/m2. Isopycnic analysis of repaired deoxyribonucleic acid from this strain revealed a two-fold increase over its wild-type counterpart in the amount of repair replication performed after a dose of 15 J/m2. Sedimentation velocity analysis of DNA after selective photolysis of bromouracil-containing repaired regions showed that the uvrD101 mutation exerted its primary effect on the long-patch component of excision repair. The uvrD101 mutant performed long-patch repair at a larger number of sites than the number repaired by this mode in the wild-type strain; these patches were more extensive in length than the long-patch component in wild-type.

In vitro replication of bacteriophage T7 DNA damaged by ultraviolet radiation.

The effect of ultraviolet radiation on DNA replication has been examined with an in vitro system capable of replicating intact chromosomes of T7 DNA from an exogenous template. Exposure of the template DNA to ultraviolet radiation resulted in a sharp drop in the amount of in vitro DNA synthesis. The residual replication detected when irradiated templates were used was found to proceed semiconservatively and to result in the production of pieces of duplex DNA approximately the same size as the average distance between pyrimidine dimers. It was also found that prior irradiation of the template inhibits formation of fast-sedimenting concatemer-like DNA structures normally synthesized in vitro. Hybridization studies demonstrated that the product synthesized in vitro from ultraviolet-irradiated templates includes DNA from both the left and right halves of the T7 chromosome. This may mean that after ultraviolet irradiation more than one origin of replication exists.

Effect of the uvrD mutation on excision repair.

A pair of related Escherichia coli K-12 strains, one of which contains the uvrD101 mutation, were constructed and compared for ability to perform various steps in the excision repair of deoxyribonucleic acid damage inflicted by ultraviolet radiation. The results of this study indicated: (i) ultraviolet sensitivity in the uvrD101 mutant was greater than that of wild type but less than that measured in an incision-deficient uvrA mutant; (ii) host cell reactivation paralleled the survival data; (iii) postirradiation deoxyribonucleic acid degradation was virtually identical in the two strains; (iv) incision, presumably at the sites of pyrimidine dimers, proceeded normally in the uvrD101 strain; (v) excision of pyrimidine dimers was markedly reduced in both rate and extent in the uvrD101 mutant; (vi) the amount of repair resynthesis was the same in both strains, and there was no evidence of abnormally long repair patches in the uvrD mutant; and (vii) rejoining of incision breaks was slow and incomplete in the uvrD strain. These data suggest that the ultraviolet sensitivity conferred by the uvrD mutation arises from inefficient removal of pyrimidine dimers or from failure to close incision breaks. The data are compatible with the notion that the uvrD+ gene produce affects the conformation of incised deoxyribonucleic acid molecules.

In vitro recombination of bacteriophage T7 DNA damaged by UV radiation.

A system capable of in vitro packaging of exogenous bacteriophage T7 DNA has been used to monitor the biological activity of DNA replicated in vitro. This system has been used to follow the effects of UV radiation on in vitro replication and recombination. During the in vitro replication process, a considerable exchange of genetic information occurs between T7 DNA molecules present in the reaction mixture. This in vitro recombination is reflected in the genotype of the T7 phage produced after in vitro encapsulation; depending on the genetic markers selected, recombinants can comprise nearly 20% of the total phage production. When UV-irradiated DNA is incubated in this system, the amount of in vitro synthesis is reduced and the total amount of viable phage produced after in vitro packaging is diminished. In vitro recombination rates are also lower when the participating DNA molecules have been exposed to UV. However, biochemical and genetic measurements confirmed that there is little or no transfer of pyrimidine dimers from irradiated DNA into undamaged molecules.