UV-induced base substitution mutations in a shuttle vector plasmid propagated in group C xeroderma pigmentosum cells.

To assess the contribution to mutagenesis of human DNA repair defects, the UV-irradiated shuttle vector plasmid pZ189 was propagated in fibroblasts derived from a xeroderma pigmentosum (XP) patient in DNA repair complementation group C. In comparison to results with DNA repair-proficient human cells (WI-38 VA13), UV-irradiated pZ189 propagated in the XP-C (XP4PA(SV)) cells showed fewer surviving plasmids and a higher frequency of mutated plasmids. Base sequence analysis of 67 mutated plasmids recovered from the XP-C cells revealed similar classes of point mutations and mutation spectrum, and a higher frequency of G:C to A:T transitions along with a lower frequency of transversions among plasmids with single or tandem mutations compared to plasmids recovered from the normal line. Most single-base substitution mutations (83%) occurred at G:C base pairs in which the 5'-adjacent base of the cytosine was thymine or cytosine. These results indicate that the DNA repair defects in XP-C, in comparison to data previously reported for XP-A, XP-D and XP-F, result in different UV survival and mutation frequency but in similar types of base substitution mutations.     

Mutational hotspot variability in an ultraviolet-treated shuttle vector plasmid propagated in xeroderma pigmentosum and normal human lymphoblasts and fibroblasts

The mutagenesis shuttle vector, pZ189, was treated with ultraviolet (u.v.) radiation in vitro and passed through a DNA repair-deficient lymphoblastoid cell line derived from a patient with xeroderma pigmentosum complementation group A (XP-A) (XP12BE(EBV)) and a DNA repair-proficient lymphoblastoid cell line (GM606(EBV)). After u.v. treatment, plasmid survival was lower and mutation frequency higher with the XP-A cells mirroring the survival and mutagenesis of the host cells. The nature of the mutations in the suppressor tRNA marker gene was determined by direct sequence analysis. The G.C to A.T transition was the dominant (85%) base substitution mutation with the XP lymphoblasts and was the major (56%) base substitution mutation with the repair-proficient lymphoblasts. We found a G.C to A.T transition mutational hotspot with the XP lymphoblasts not seen in our previous experiments with fibroblasts from the same patient. Comparison of the data presented here with our results with DNA repair-deficient and DNA repair-proficient fibroblasts suggests that hotspot variability is not due to genetic polymorphism or repair capacity of the cells. Instead it appears that cellular factors can influence the probability of mutagenesis of modified DNA at particular sites.     

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.     

Nuclear deoxyribonuclease activities in normal and xeroderma pigmentosum lymphoblastoid cells

Deoxyribonuclease activities were examined in isoelectric focusing fractions of non-histone chromatin-associated and nucleoplasmic proteins of isolated nuclei of normal human and xeroderma pigmentosum, complementation group A, lymphoblastoid cells using parallel procedures. In the nucleoplasm of both cell lines, a very similar series of both DNA endo- and exo-nuclease activities were found; in chromatin a series of similar endonuclease but no exonuclease activites were present. Several differences were observed in the xeroderma pigmentosum cells, however, notably a striking increase in DNA endonuclease activity in a chromatin fraction at pI 4.6 against linear duplex DNA and a decrease in a chromatin endonuclease activity focusing at pI 7.8.     

Comparison of histones in normal and xeroderma pigmentosum lymphoblastoid cells

Histones from normal human and xeroderma pigmentosum, complementation group A (XPA), lymphoblastoid cells were compared both quantitatively, qualitatively and for binding affinity for DNA. Electrophoretic examination of the histones showed that all five major histone species were present in both cell groups and that there were no quantitative differences between normal and XPA histones. Binding affinity to [3H] mammalian DNA of the histones was determined. No significant differences were observed in binding of either normal or XPA histones to DNA.     

DNA-binding proteins in xeroderma pigmentosum fibroblasts.

DNA-binding proteins in human fibroblasts were examined by chromatography on DNA-cellulose columns. By successive chromatography on columns containing native, denatured, and UV-irradiated DNA-cellulose respectively the proteins binding to different types of DNA could be studied. Elution of the columns with sodium chloride followed by polyacrylamide gel electrophoresis allowed several DNA-binding proteins to be identified. All of the major DNA-binding proteins were present in strains of xeroderma pigmentosum cells respectively deficient in excision-repair and post-replication repair of ultraviolet-induced damage.     

Transformation and immortalization of diploid xeroderma pigmentosum fibroblasts

Diploid xeroderma pigmentosum (XP) skin fibroblast strains from various XP-complementation groups (B, C, G, and H) were transformed with an origin-defective SV40 early region or with the pSV3 gpt plasmid. In the latter case, transfected cells were selected for their ability to express the dominant xgpt gene. Immortalized cell lines were obtained, from XP-complementation groups C (8CA, 3MA, and 20MA; XP3MA and XP20MA were formerly considered to belong to complementation group I), G (2BI and 3BR), and H (2CS). No immortalized cells could be isolated from complementation group B (11BE). The immortalization frequency of wild-type diploid fibroblasts and diploid cultures from XP patients was not significantly increased by cotransfection with the SV40 early region plus several selected viral and cellular oncogenes. In fact, co-transfection with some of the oncogenes caused a marked decrease of the transformation frequency. The observed immortalization occurred at a frequency of approximately 5 x 10(-8).     

Ultraviolet light-resistant primary transfectants of xeroderma pigmentosum cells are also DNA repair-proficient

In a previous work, an immortal xeroderma pigmentosum cell line belonging to complementation group C was complemented to a UV-resistant phenotype by transfection with a human cDNA clone library. We now report that the primary transformants selected for UV-resistance also acquired normal levels of DNA repair. This was assessed both by measurement of UV-induced [3H]thymidine incorporation and by equilibrium sedimentation analysis of repair-DNA synthesis. Therefore, the transduced DNA element which confers normal UV-resistance also corrects the excision repair defect of the xeroderma pigmentosum group C cell line.     

Modulation of an ultraviolet mutational hotspot in a shuttle vector Xeroderma cells.

Ultraviolet mutagenesis of the shuttle vector plasmid pZ189 in Xeroderma Pigmentosum cells yields a mutational pattern marked by hotspots at photoproduct sites on both strands of the supF marker gene. In order to test the influence of strand orientation on the appearance of hotspots the mutagenesis study was repeated on a vector with the supF gene in the inverted orientation. We recovered a pattern the same as that in the earlier work and conclude that the nature of the DNA polymerase involved in the replication of specific strands is not a primary determinant of hotspot occurrence in this system. One of the hotspots lies in an 8 base palindrome while the corresponding site on the other strand was not a hotspot. These results were obtained with calcium phosphate transfection of the UV treated vector. When DEAE dextran was used as a transfection agent both sites in the palindrome were hotspots. In a mixing experiment the calcium phosphate pattern was recovered. Our data suggest that the sequence determinants of mutational probability at these two sites lie outside the 8 bases of the palindrome and that mutagenesis at one, but not the other, site is sensitive to perturbation of cellular calcium levels.     

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).     

Increased sensitivity of a xeroderma pigmentosum lymphoblastoid cell line to serum deprivation in vitro

Summary Two human lymphoblastoid cell lines, GM 1989, from a normal individual, and GM 2345, from a patient with xeroderma pigmentosum, complemetation group A, were selected for comparative biochemical studies because they both grow rapidly and at vitually identical rates in sealed flasks in RPMI 1640 medium buffered to physiological pH with HEPES buffer, supplemented with 12% heat-inactivated fetal bovine serum. Although the two cell lines showed no difference in growth parameters assayed by standard methods, further studies showed that the GM 2345 cell line was markedly more sensitive to diminution of the serum concentration of the culture medium than was the normal cell line. These results indicate that lymphoblastoid cell lines, particularly those from individuals with certain genetic or metabolic diseases, may be growing under marginal or limiting circumstances, different from those of control cell lines, which are not detected by standard techniques used to monitor mammalian cell cultures. Supported by Basil O'Connor Grant 5-287 from the March of Dimes Birth Defects Foundation, the Dermatology Foundation, and the Foundation of the University of Medicine and Dentistry of New Jersey.     

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.     

Excision repair in xeroderma pigmentosum group C cells is regulated differently in transformed cells and primary fibroblasts

Excision repair in xeroderma pigmentosum group C cells occurs at about 20-30% of normal levels. In confluent fibroblasts a unique characteristic of this low repair is that it is clustered, representing very efficient repair in a small region of the genome. In SV40-transformed fibroblasts and Epstein-Barr virus-transformed lymphocytes of complementation group C, however, excision repair is randomly distributed. This may be a consequence of the high rate of proliferation of both of these cell types, because random repair is also observed in rapidly proliferating group C fibroblasts. The distribution of sites that can be mended in group C cells, therefore, varies according to the transformed and proliferative state of the cells, demonstrating that transformed cells do not always exhibit repair characteristics identical to those of primary fibroblasts.     

Genetic complementation between UV-sensitive CHO mutants and xeroderma pigmentosum fibroblasts

The purpose of this study was to determine the feasibility of doing complementation analysis between DNA-repair mutants of CHO cells and human fibroblasts based on the recovery of hybrid cells resistant to DNA damage. Two UV-sensitive CHO mutant lines, UV20 and UV41, which belong to different genetic complementation groups, were fused with fibroblasts of xeroderma pigmentosum in various complementation groups. Selection for complementing hybrids was performed using a combination of ouabain to kill the XP cells and mitomycin C to kill the CHO mutants. Because the frequency of viable hybrid clones was generally < 10⁻⁶ and the frequency of revertants of each CHO mutant was 2×10⁻⁷, putative hybrids required verification. The hybrid character of clones was established by testing for the presence of human DNA in a dot-blot procedure.

Hybrid clones were obtained from 9 of the 10 different crosses involving 5 complementation groups of XP cells. The 4 attempted crosses with 2 other XP groups yielded no hybrid colonies. Thus, a definitive complementation analysis was not possible. Hybrids were evaluated for their UV resistance using a rapid assay that measures differential cytotoxicity (DC). All 9 hybrids were more resistant than the parental mutant CHO and XP cells, indicating that in each case complementation of the CHO repair defect by a human gene had occurred. 3 hybrids were analyzed for their UV-radiation survival curves and shown to be much more resistant that the CHO mutants but less resistant than normal CHO cells. With 2 of these hybrids, sensitive subclones, which had presumably lost the complementing gene, were found to have similar sensitivity to the parental CHO mutants. We conclude that the extremely low frequency of viable hybrids in this system limits the usefulness of the approach. The possibility remains that each of the nonhybridizing XP strains could be altered in the same locus as one of the CHO mutants.     

Expression of a mammalian DNA photolyase confers light-dependent repair activity and reduces mutations of UV-irradiated shuttle vectors in xeroderma pigmentosum cells

Photoreactivation is one of the DNA repair mechanisms to remove UV lesions from cellular DNA with a function of the DNA photolyase and visible light. Two types of photolyase specific for cyclobutane pyrimidine dimers (CPD) and for pyrimidine (6-4) pyrimidones (6-4PD) are found in nature, but neither is present in cells from placental mammals. To investigate the effect of the CPD-specific photolyase on killing and mutations induced by UV, we expressed a marsupial DNA photolyase in DNA repair-deficient group A xeroderma pigmentosum (XP-A) cells. Expression of the photolyase and visible light irradiation removed CPD from cellular DNA and elevated survival of the UV-irradiated XP-A cells, and also reduced mutation frequencies of UV-irradiated shuttle vector plasmids replicating in XP-A cells. The survival of UV-irradiated cells and mutation frequencies of UV-irradiated plasmids were not completely restored to the unirradiated levels by the removal of CPD. These results suggest that both CPD and other UV damage, probably 6-4PD, can lead to cell killing and mutations.     

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.     

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.     

Nuclear DNA endonuclease activities on partially apurinic/apyrimidinic DNA in normal human and xeroderma pigmentosum lymphoblastoid and mouse melanoma cells

DNA endonuclease activities from nuclear proteins of normal human and xeroderma pigmentosum (XP), complementation group A, lymphoblastoid and Cloudman mouse melanoma cells were examined against partially apurinic/apyrimidinic (AP) DNA. Non-histone chromatin-associated and nucleoplasmic proteins, obtained from isolated nuclei, were subfractionated by isoelectric focusing and assayed for DNA endonuclease activity against linear, calf thymus DNA. All of the nine chromatin-associated and three of the nucleoplasmic fractions, which lacked DNA exonuclease activity, were tested for DNA endonuclease activity against both native and partially AP, circular, duplex, supercoiled PM2 DNA. In all three cell lines, four chromatin-associated, but none of the nucleoplasmic fractions, showed increased activity against DNA rendered AP by either heat/acid treatment or by alkylation with methyl methanesulfonate (MMS) followed by heat. One chromatin-associated activity, with pI 9.8, which was not active on native DNA, showed the greatest activity on AP DNA. AP activity was moderately decreased in XP cells and slightly decreased in mouse melanoma cells, as compared with normal cells, in the fraction at pI 9.8. Little or no increased activity was observed in any of the endonucleases from any of the cell lines on MMS alkylated 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.