Complementing xeroderma pigmentosum fibroblasts restore biological activity to UV-damaged DNA.

UV survival curves of adenovirus 2 using fused, complementing xeroderma pigmentosum (XP) fibroblast strains as virus hosts showed a component with an inactivation slope identical to that given by normal cells. This component was not observed when the fibroblasts were not fused or when fusion involved strains in the same complementation group. Extrapolation of this component indicated that at zero dose 3% of the viral plaque-forming units had infected cells capable of normal repair. These results suggest that 3% of the cells were complementing heterokaryons, a value similar to that actually observed by autoradiographic analysis of UV-induced unscheduled DNA synthesis. Thus, heterokaryons formed from XP fibroblasts belonging to different complementation groups are as capable of restoring biological activity to UV-damaged adenovirus 2 as are normal cells.     

Fluorescent-light-induced lethality and DNA repair in normal and xeroderma pigmentosum fibroblasts

Cell survival and induction of endonuclease-sensitive sites in DNA were measured in human fibroblast cells exposed to fluorescent light or germicidal ultraviolet light. Cells from a xeroderma pigmentosum patient were hypersensitive to cell killing by fluorescent light, although less so than for germicidal ultraviolet light. Xeroderma pigmentosum cells were deficient in the removal of fluorescent light-induced endonuclease sites that are probably pyrimidine dimers, and both the xeroderma pigmentosum and normal cells removed these sites with kinetics indistinguishable from those for ultraviolet light-induced sites. A comparison of fluorescent with ultraviolet light data demonstrates that there are markedly fewer pyrimidine dimers per lethal event for fluorescent than for ultraviolet light, suggesting a major role for non-dimer damage in fluorescent light lethality.     

DNA-protein cross-linking by ultraviolet radiation in normal human and xeroderma pigmentosum fibroblasts.

DNA-protein cross-linking by ultraviolet radiation was measured in human fibroblasts by an adaptation of the method of DNA alkaline elution. To measure cross-linking, a controlled frequency of DNA single-strand breaks was introduced by exposing the cells to a low dose of X-ray at 0 degrees C prior to analysis by alkaline elution. The effect of prior exposure of the cells to ultraviolet radiation was to reduce the rate and/or extent of DNA elution from X-irradiated cells. This effect was attributed to DNA-protein cross-linking, since the effect was reversed by treatment of the cell lysates with proteinase-K. Cross-linking in normal human fibroblasts occurred immediately after ultraviolet irradiation, prior to the appearance of DNA single-strand breaks due to excision repair. Upon incubation of normal cells after exposure, to ultraviolet radiation, the cross-linking was partially repaired. In xeroderma pigmentosum cells, cross-links appeared as in normal cells, but there was no repair. Instead, the extent of cross-linking appeared to increase upon incubation after ultraviolet irradiation.     

Levels of DNA polymerase-alpha and beta in normal and xeroderma pigmentosum fibroblasts.

We have determined the levels of DNA-polymerases-alpha and-beta in fibroblasts obtained from normal subjects and from patients with Xeroderma Pigmentosum (XP) belonging to three different complementation groups and to the variant form. The assays have been performed in crude extracts and after fractionation on sucrose gradients. The levels of alpha and beta-polymerases in the different cases of XP were found to lie within the same range as the control values, and no correlation was found with the severity of the symptoms. The sedimentation coefficients of the two polymerases from all the pathological lines were identical to those of the normal fibroblasts.     

The induction of DNA strand breaks in normal human skin fibroblasts exposed to solar ultraviolet radiation

The levels of apparent DNA single-strand breaks (ssb) were measured, following a 0-20 h incubation of normal human skin fibroblasts exposed to the solar uv wavelengths produced by a fluorescent sunlamp. The ssb were determined using the alkaline elution assay, which was performed either without proteinase K (proK) or in its presence, so as to eliminate any DNA-protein crosslinks that may be present in the cells. Cells were irradiated with either 3 kJ/m2 of sunlamp uv greater than 295 nm, 150 kJ/m2 of sunlamp uv greater than 315 nm, or 150 kJ/m2 of sunlamp uv greater than 320 nm. These treatments resulted in the production of 5-10 ssb/10(10) Da. For the two shorter wavelength irradiations, the levels of ssb decreased rapidly upon incubation of the cells. However, when the elutions were performed using proK, the number of ssb increased about twofold following a 2-4 h incubation. In contrast, the levels of ssb decreased in the sunlamp uv greater than 320 nm irradiated cells for elutions performed with or without proK. These results suggest that under certain irradiation conditions, ssb are formed in cells upon incubation, which are hidden by the crosslinking of protein to DNA.     

Replication of simian virus 40 DNA in normal human fibroblasts and in fibroblasts from xeroderma pigmentosum.

Simian virus 40 infection of semipermissive human diploid fibroblasts (HF), at early passage in cell culture, was compared with that of permissive established monkey cell lines. Viral DNA can be readily detected at 24 to 48 h postinfection at 37 degrees C with a high multiplicity of infection, approaching 10% of that of monkey cells (TC7). The length of time necessary for replication of an average molecule of viral DNA was found to be indistinguishable in HF and TC7 cells. Strand elongation plus termination were assessed by following the accumulation of DNA I at 40 degrees C from replicative intermediates of tsA30 prelabeled at 33 degrees C, obviating isotope pool problems. Combined initiation and elongation of wild-type viral DNA was measured by density shift experiments involving a 5-bromodeoxyuridine chase of prelabeled [3H]thymidine-labeled viral DNA. Determination of accumulation of viral T and V antigens supports the conclusion that the most likely basis for the reduced virus yield in HF cells results from the inefficiency of an early stage in virus infection, before or during uncoating. Similar results were obtained in fibroblasts derived from patients with xeroderma pigmentosum, suggesting that enzymes of UV repair are not required in unirradiated simian virus 40 DNA synthesis.     

Semiautomated autoradiographic measurement of DNA repair in normal and xeroderma pigmentosum cultured human fibroblasts

Summary Assessment of DNA repair in cultured human fibroblasts by autoradiography may be facilitated by using semiautomated grain counting instruments. The instrument-determined number of autoradiographic grains per nucleus in cultured human skin fibroblasts was found to be linear in comparison to visual counts up to only 30 grains per nucleus. However, with two different instruments a greater range of linearity (to 100 to 120 grains per nucleus) was attained by measuring the grain surface area per nucleus. Semiautomated analysis of the grain surface area per nucleus yielded measurements of relative rates of unscheduled DNA synthesis after ultraviolet irradiation in xeroderma pigmentosum and normal human fibroblasts, which were reproducible and rapid.     

Induction and repair of psoralen cross-links in DNA of normal human and xeroderma pigmentosum fibroblasts

Skin fibroblasts from normal human subjects were exposed in vitro to long-wave ultraviolet radiation (UVA, 320–400 nm) alone, or in combination with 8-methoxypsoralen (8-MOP). DNA damage was analysed with the alkaline elution technique before and after post-treatment incubation of the cells at 37°C for various times.Cells treated with UVA at 1.1 J/cm² showed an increased DNA elution rate, which returned to the normal level within 30 min of post-treatment incubation. In cells treated with PUVA (8-MOP at 20 μg/ml plus UVA at 0.04 J/cm²), the alkaline elution rate was not different from untreated control cells, either before or after post-treatment incubation for times up to 7 days.When the PUVA treatment was followed first by a washing, to remove any unbound 8-MOP, and then by UVA (PUVA + UVA) at 1.1 J/cm², the alkaline elution rate decreased below the control level. During the post-treatment incubation of the PUVA + UVA-treated cells there was a gradual increase of the alkaline elution rate to a level significantly above that in control cells. This increase was observed after 30 min. It reached a miaximum after 24 h and remained after 7 days of post-treatment incubation. Cells from a patient with xeroderma pigmentosum of complementation group A, which were given the same PUVA + UVA treatment, did not show any change in the alkaline elution rate during the post-treatment incubation.If, as seems likely, an increased alkaline elution rate indicates an increase of DNA breaks, and a decreased alkaline elution rate indicates the sealing of breaks and/or the formation of cross-links, the results would suggest the following: (1) UVA irradiation in itself is capable of inducing DNA breaks, which are rapidly sealed during post-treatment incubation; (2) PUVA treatment induces mono-adducts, some of which appear to remain in the DNA for at least 7 days of post-treatment incubation and can be activated to form DNA cross-links by a second dose of UVA; (3) DNA cross-links induced by PUVA + UVA can be recognized by a repair process that involves the formation of DNA breaks. This process is not observed in xeroderma pigmentosum cells of group A.     

Enhanced expression of procollagenase in ataxia-telangiectasia and xeroderma pigmentosum fibroblasts

Summary Ataxia-telangiectasia and xeroderma pigmentosum are human hereditary diseases in which patients are cancer prone and demonstrate increased sensitivity to DNA damage by ionizing and ultraviolet radiation, respectively. In culture, both ataxia-telangiectasia and xeroderma pigmentosum skin fibroblasts show increased synthesis and secretion of the extracellular matrix proteins fibronectin and collagen. To determine whether these differences in protein production result from fundamental abnormalities in regulation of genes associated with cellular interactions, we compared the effects of trifluoperazine and 12-O-tetradecanoylphorbol-13-acetate on expression of the extracellular matrix-degrading metalloproteinases, procollagenase and prostromelysin, by normal, ataxia-telangiectasia, and xeroderma pigmentosum fibroblasts. After trifluoperazine treatment the overall levels of these metalloproteinases were much greater in three ataxia-telangiectasia cell strains and in cells from xeroderma pigmentosum complementation groups A and D than in normal cells. In contrast, cells from xeroderma pigmentosum complementation group C produced only slightly more procollagenase than normal cells. 12-O-tetradecanoylphorbol-13-acetate also induced higher than normal levels of procollagenase in some ataxia-telangiectasia and xeroderma pigmentosum strains, but less than that induced by trifluoperazine. Because increased extracellular accumulation of matrix-degrading enzymes has long been implicated in metastatic progression, this altered expression of procollagenase and prostromelysin in ataxia-telangiectasia and xeroderma pigmentosum cells could play an important role in the pathogenesis of various tumors in individuals with these genetic diseases. This work was supported by the Office of Health and Environmental Research, U. S. Department of Energy (contract DE-AC03-76-SF01012) (J. A., J. P. M.) and by a Fellowship in Medical Research from the A. P. Giannini/Bank of America Foundation (J. A.). A summary of these results has appeared previously in abstract form (1).     

Alterations in levels of 5'-adenyl dinucleotides following DNA damage in normal human fibroblasts and fibroblasts derived from patients with xeroderma pigmentosum

Levels of 5'-adenyl dinucleotides, measured as diadenosine-5',5'-P1,P4-tetraphosphate (Ap4A), were found to accumulate in cultured human fibroblasts following treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), the radiomimetic drug bleomycin, and nitroquinoline-1-oxide (NQO) or UV-irradiation in the presence of cytosine arabinofuranoside (araC). In contrast, cells derived from patients with xeroderma pigmentosum complementation group A (XP-A) did not demonstrate an increase in DNA-strand breaks following UV irradiation or NQO in the presence of araC nor an increase in Ap4A levels. Ap4A accumulation did occur in XP-A cells following treatment with MNNG. Cells derived from patients characterized as XP variants, which are incision repair-proficient, accumulated 5'-dinucleotides following bleomycin, MNNG and UV or NQO in the presence of araC. Taken together, these data suggest that Ap4A accumulates as a response to DNA-strand breaks.     

Ultraviolet mutagenesis of normal and xeroderma pigmentosum variant human fibroblasts

The mutabilities of normal and xeroderma pigmentosum variant (XP4BE) human fibroblasts by ultraviolet light (UV) were compared under conditions of maximum expression of the 6-thioguanine resistance (TGr) phenotype. Selection was with 20 micrograms TG/ml on populations reseeded at various times after irradiation. Approx. 6--12 days (4--8 population doublings), depending on the UV dose, were necessary for complete expression. The induced mutation frequencies were linear functions of the UV dose but the slope of the line for normal cells extrapolated to zero induced mutants at 3 J/m2. The postreplication repair-defective XP4BE cells showed a higher frequency of TGr colonies than normal fibroblasts when compared at equal UV doses or at equitoxic treatments. The induced frequency of TGr colonies was not a linear function of the logarithm of survival for either cell type. Instead, the initial slope decreased to a constant slope for survivals less than about 50%. The UV doses and induced mutation frequencies corresponding to 37% survival of cloning abilities were 6.7 J/m2 and 6.2 X 10(-5), respectively, for normal cells and 3.75 J/m2 and 17.3 X 10(-5) for the XP4BE cells. The lack of an observable increase in the mutant frequency for normal fibroblasts exposed to slightly lethal UV doses suggests that normal postreplication repair of UV-induced lesions is error-free (or nearly so) until a threshold dose is exceeded.     

Electrodeless dielectrophoresis of single- and double-stranded DNA

Dielectrophoretic trapping of molecules is typically carried out using metal electrodes to provide high field gradients. In this paper we demonstrate dielectrophoretic trapping using insulating constrictions at far lower frequencies than are feasible with metallic trapping structures because of water electrolysis. We demonstrate that electrodeless dielectrophoresis (EDEP) can be used for concentration and patterning of both single-strand and double-strand DNA. A possible mechanism for DNA polarization in ionic solution is discussed based on the frequency, viscosity, and field dependence of the observed trapping force.     

Single strand DNA breaks in rat brain cells exposed to microwave radiation

This investigation concerns with the effect of low intensity microwave (2.45 and 16.5 GHz, SAR 1.0 and 2.01 W/kg, respectively) radiation on developing rat brain. Wistar rats (35 days old, male, six rats in each group) were selected for this study. These animals were exposed for 35 days at the above mentioned frequencies separately in two different exposure systems. After the exposure period, the rats were sacrificed and the whole brain tissue was dissected and used for study of single strand DNA breaks by micro gel electrophoresis (comet assay). Single strand DNA breaks were measured as tail length of comet. Fifty cells from each slide and two slides per animal were observed. One-way ANOVA method was adopted for statistical analysis. This study shows that the chronic exposure to these radiations cause statistically significant (p<0.001) increase in DNA single strand breaks in brain cells of rat.     

Characterization of ciprofloxacin binding to the linear single- and double-stranded DNA

The binding of ciprofloxacin to natural and synthetic polymeric DNAs was investigated at different solvent conditions using a combination of spectroscopic and hydrodynamic techniques. In 10 mM cacodylate buffer (pH 7.0) containing 108.6 mM Na(+), no sequence preferences in the interaction of ciprofloxacin with DNA was detected, while in 2 mM cacodylate buffer (pH 7.0) containing only 1.7 mM Na(+), a significant binding of ciprofloxacin to natural and synthetic linear double-stranded DNA was observed. At low ionic strength of solution, ciprofloxacin binding to DNA duplex containing alternating AT base pairs is accompanied by the largest enhancement in thermal stability (e.g. DeltaT(m) approximately 10 degrees C for poly[d(AT)].poly[d(AT)]), and the most pronounced red shift in the position of the maximum of the fluorescence emission spectrum (lambda(max)). Similar red shift in the position of lambda(max) is also observed for ciprofloxacin binding to dodecameric duplex containing five successive alternating AT base pairs in the row. On the other hand, ciprofloxacin binding to poly[d(GC)].poly[d(GC)], calf thymus DNA and dodecameric duplex containing a mixed sequence is accompanied by the largest fluorescence intensity quenching. Addition of NaCl does not completely displace ciprofloxacin bound to DNA, indicating the binding is not entirely electrostatic in origin. The intrinsic viscosity data suggest some degree of ciprofloxacin intercalation into duplex.     

DNA damage and repair in Chinese hamster fibroblasts exposed to gamma and secondary radiation generated by 70 GeV protons

The cytochemical study of DNA damage and repair in a Chinese hamster fibroblast culture exposed to gamma-rays and secondary radiation from 70 GeV protons showed no significant differences between the two types of radiation.     

The inhibition of DNA repair by aphidicolin or cytosine arabinoside in X-irradiated normal and xeroderma pigmentosum fibroblasts

Normal and excision-deficient xeroderma pigmentosum fibroblasts were X-irradiated and the influence on DNA repair of either the repair inhibitor cytosine arabinoside or the specific inhibitor of Dna polymerase alpha, aphidicolin, investigated. The data indicated that the repair of a certain fraction of X-ray-induced lesions can be inhibited in both cell lines by both compounds. Thus, as aphidicolin blocks the operation of polymerase alpha, this enzyme must be involved in an excision repair pathway operating in both normal and excision-deficient xeroderma pigmentosum cells.