The repair of UV-irradiated plasmids transfected into cultured fish cells

UV-irradiated plasmids (pNPV B and pBSCATSV) were transfected into RBCF-1 cells derived from a goldfish (Carassius auratus) and into a xeroderma pigmentosum (group A) cell line, XP20SSV. The frequency of stable neor transformation by pNPV B decreased in a dose-dependent manner. However, in spite of large differences in UV sensitivity detected in the colony formation assay, the dose-response curves of RBCF-1 cells and XP20SSV cells were almost the same. The photorecovery (PR) of transforming activity of UV-irradiated plasmids was confirmed in RBCF-1 cells but its extent was much smaller than that observed in the survival assay. The expression of the transfected cat (chloramphenicol acetyltransferase; CAT) gene after 24-h incubation in the dark was much more sensitive to UV irradiation when compared with the stable transformation assay. The extent of PR of cat gene expression in RBCF-1 cells was high and comparable with that of the survival assay. The CAT value of RBCF-1 cells transfected with UV-irradiated plasmids relative to that of unirradiated controls increased as incubation time in the dark after transfection became longer. This suggests that the UV lesions on the plasmids transfected in the RBCF-1 cells were repaired in the dark. The cat gene expression of UV-irradiated plasmids in XP20SSV was very low and independent of incubation time after transfection.     

v-myb transformation of Xeroderma pigmentosum human fibroblasts: overexpression of the c-Ha-ras oncogene in the transformed cells

Human Xeroderma pigmentosum "normal" fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental "normal" AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.     

Identification of a phosphoprotein specifically induced by the transforming DNA of rat neuroblastomas

DNAs from nitrosoethylurea-induced rat neuroblastomas transform NIH/3T3 mouse fibroblasts in a transfection assay. DNAs of such transformed cells can be used in a subsequent cycle of transfection to generate secondary foci that contain virtually no foreign genetic material besides the sequences carrying the rat neuroblastoma transforming function. These secondary neuroblastoma transfectants were injected into young mice and grew out into fibrosarcomas. Sera from these mice were examined for reactivity with any proteins which were induced specifically by the neuroblastoma transforming sequence. These sera precipitate a polypeptide of about 185,000 daltons from ³⁵S-methionine-labeled cell lysates of the rat neuroblastoma cells that served as DNA donors and in all transfection-derived primary and secondary foci. This protein is present in high levels in all neuroblastoma transfectant clones, but was not detectable in a variety of other transformed cells. Antisera were prepared from mice bearing tumors induced by transformed cells derived by transfection of DNAs from various tumor cell types unrelated to rat neuroblastoma. These antisera failed to immunoprecipitate the 185,000 dalton protein. These data indicate that the synthesis of the 185,000 dalton protein is specifically induced by the neuroblastoma transforming sequence. The protein may be encoded by the transforming sequence and may mediate transformation in this chemically induced tumor.     

Isolation and partial characterization of virus-transformed cell lines representing the A, G and variant complementation groups of xeroderma pigmentosum

We have established viral-transformed, apparently permanent (immortalized) cell lines from diploid fibroblasts representative of normal and xeroderma pigmentosum (XP) A, G and variant individuals. The XP-G and XP-variant cells represent complementation groups not previously available as permanent lines. All the new permanent cell lines exhibit SV40 T-antigen expression. They are also aneuploid and have growth characteristics typical of viral transformants. They have retained the phenotypes of UV sensitivity, reduced repair synthesis or defective 'postreplication repair' appropriate to the XP complementation group they represent. Additionally, the new cell lines are all transfectable with the selectable plasmid pRSVneo. The XP-G and XP-variant cell lines show enhanced transfection with UV-irradiated plasmid DNA; a phenomenon previously reported for normal immortalized cells and for immortalized cells from the A and F complementation groups of XP.     

Host-cell reactivation of UV-irradiated and chemically-treated herpes simplex virus-1 by xeroderma pigmentosum, XP heterozygotes and normal skin fibroblasts

The host-cell reactivation of UV-irradiated and N-acetoxy-2-acetylamino-fluorene-treated herpes simplex virus type 1 strain MP was studied in normal and xeroderma pigmentosum human skin fibroblasts. Virus treated with either agent demonstrated lower survival in XP cells from complementation groups A, B, C and D than in normal fibroblasts. The relative reactivation ability of XP cells from the different genetic complementation groups was found to be the same for both irradiated and chemically treated virus. In addition, the inactivation kinetics for virus treated with either agent in the XP variant were comparable to that seen in normal skin fibroblasts. The addition of 2 or 4 mmoles caffeine to the post-infection assay medium had no effect on the inactivation kinetics of virus treated by either agent in the XP variant or in XP cells from the different genetic complementation groups. Treatment of the virus with nitrogen mustard resulted in equivalent survival in normal and XP genetic complementation group D cells. No apparent defect was observed in the ability of XP heterozygous skin fibroblasts to repair virus damaged with up to 100 μg N-acetoxy-2-acetylaminofluorene per ml. These findings indicate that the repair of UV-irradiated and N-acetoxy-2-acetylaminofluorene-treated virus is accomplished by the same pathway or different pathways sharing a common intermediate step and that the excision defect of XP cells plays little if any role in the reactivation of nitrogen mustard treated virus.     

Postreplication repair of DNA in human fibroblasts after UV irradiation or treatment with metabolites of benzo(a)pyrene

We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.     

Activation of a novel human transforming gene, ret, by DNA rearrangement

A novel transforming gene was detected by transfection of NIH 3T3 cells with human lymphoma DNA. The tumor DNA induced a single focus in primary transfections, whereas DNAs of transformed NIH cells induced transformation with high efficiencies in secondary and tertiary assays. Molecular clones spanning about 37 kb of human sequence were isolated from tertiary transformant DNA. Blot hybridization indicated that the transforming gene consisted of two segments that were unlinked in both normal human and primary lymphoma DNAs. The two segments of human DNA were cotranscribed in transformed NIH cells but not in any human cells examined. The transforming gene thus appeared to be activated by recombination between two unlinked human DNA segments, possibly by cointegration during transfection.     

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.     

U.V. enhanced reactivation of U.V.-and gamma-irradiated adenovirus in Cockayne syndrome and Xeroderma pigmentosum fibroblasts

U.V.-enhanced reactivation (UVER) of both U.V.-irradiated and gamma-irradiated human adenovirus type 2 (Ad 2) was examined following the infection of a variety of Cockayne Syndrome (CS) and Xeroderma pigmentosum (XP) fibroblast strains which had been pre-irradiated with U.V. light. U.V.-irradiated or non-irradiated fibroblasts were infected with either non-irradiated or irradiated Ad2, and at 48 hours after infection cells were examined for the presence of viral structural antigens (Vag) using immunofluorescent staining. Normal levels of UVER (i.e. 2-4 fold) of U.V.- and of gamma-irradiated Ad 2 were detected in 2 CS strains (CS IBE and CS 3BE), 2 XP complementation group A strains (XP 12BE and XP 25RO), and 2 XP complementation group D strains (XP 5BE and XP 6BE), although the U.V. doses to these mutant cells which resulted in peak UVER values (0 . 2 Jm-2 for XP 25RO, 0 . 14 Jm-2 for XP 12BE, 0 . 8 Jm-2 for XP 5BE and XP 6BE, and 1 . 6-5 . 0 Jm-2 for CS 1BE and CS 3BE) were considerably lower than those yielding peak UVER in normal strains (10-15 Jm-2). XP variant strains (XP 4BE and XP 115LO), however, showed substantially lower levels of UVER than normal strains.     

An immortalized xeroderma pigmentosum, group C, cell line which replicates SV40 shuttle vectors

We have established and characterized an immortalized xeroderma pigmentosum (XP), group C, cell line. Transformation of the human fibroblasts was carried out with a recombinant plasmid, pLAS-wt, containing SV40 DNA encompassing the entire early region with a defective origin of DNA replication. The transformed XP cell line, XP4PA-SVwt, and the normal transformed fibroblasts AS3-SVwt, both express SV40 T antigen together with enhanced levels of the transformation-associated cellular protein, p53. XP4PA-SVwt retains the XP UV-repair defective phenotype as demonstrated by low levels of unscheduled DNA synthesis and by the reduced survival of irradiated SV40 virus. Analysis of cellular DNA shows a single major, stable, integration site of pLAS-wt in the XP4PA-SVwt cells. The T antigen in these cells supports efficiently the replication of SV40 based shuttle vectors and should prove suitable for the introduction, expression and selection of genes related to DNA repair and to the study of mutagenesis using defined molecular probes.     

Efficient repair of 8-oxo-7,8-dihydrodeoxyguanosine in human and hamster xeroderma pigmentosum D cells

The repair of the endogenous lesion 8-oxo-7,8-dihydrodeoxyguanosine (8-oxodG) was investigated in the nucleotide excision repair mutant xeroderma pigmentosum D (XPD), using human normal or transformed XPD fibroblasts and the Chinese hamster XPD cell line UV5. In vivo repair of 8-oxodG induced by hydrogen peroxide treatment and analyzed by high-performance liquid chromatography/electrochemical detection was normal in the XPD mutant fibroblasts XP15PV and GM434, as compared to normal human fibroblasts GM970, GM5757, and GM6114. Similar results were obtained with the human SV40-transformed XPD mutant cell line GM8207 in comparison to the control cell line GM637. Repair of 8-oxodG was even slightly (2-3-fold) but reproducibly increased in Chinese hamster XPD mutant UV5 cells, as compared to parental AA8 cells. This unexpected effect was reversed by transfection in UV5 cells of a wild-type XPD cDNA and confirmed in in vitro experiments in which a plasmid substrate containing a single 8-oxoG was repaired by UV5 cell extracts. The data show that repair of 8-oxodG is normal in XPD cells, thus indicating that the neurological complications of XPD patients may not be linked to in vivo accumulation of this lesion.     

HeLa S3 DNA转染后XP细胞UV抗性的稳定增加和切除修复基因的定位探索

 通过带有选择标志的质粒pSV_2Neo的基因共转染和第一轮转化的XP细胞DNA的再转染,进一步证明了前文报道的HeLaS3 DNA的切除修复基因在XP细胞中的稳定表达,以及受体细胞UV抗性的稳定增加。通过五种限制性核酸内切酶酶切DNA片段的转染,初步寻找出切除修复基因位于Bg1 Ⅰ,xhoⅠ 酶切片段之中。     

gamma-H2AX formation in response to interstrand crosslinks requires XPF in human cells

To further define the molecular mechanisms involved in processing interstrand crosslinks, we monitored the formation of phosphorylated histone H2AX (gamma-H2AX), which is generated in chromatin near double strand break sites, following DNA damage in normal and repair-deficient human cells. Following treatment with a psoralen derivative and ultraviolet A radiation doses that produce significant numbers of crosslinks, gamma-H2AX levels in nucleotide excision repair-deficient XP-A fibroblasts (XP12RO-SV) increased to levels that were twice those observed in normal control GM637 fibroblasts. A partial XPA revertant cell line (XP129) that is proficient in crosslink removal, exhibited reduced gamma-H2AX levels that were intermediate between those of GM637 and XP-A cells. XP-F fibroblasts (XP2YO-SV and XP3YO) that are also repair-deficient exhibited gamma-H2AX levels below even control fibroblasts following treatment with psoralen and ultraviolet A radiation. Similarly, another crosslinking agent, mitomycin C, did not induce gamma-H2AX in XP-F cells, although it did induce equivalent levels of gamma-H2AX in XPA and control GM637 cells. Ectopic expression of XPF in XP-F fibroblasts restored gamma-H2AX induction following treatment with crosslinking agents. Angelicin, a furocoumarin which forms only monoadducts and not crosslinks following ultraviolet A radiation, as well as ultraviolet C radiation, resulted only in weak induction of gamma-H2AX in all cells, suggesting that the double strand breaks observed with psoralen and ultraviolet A treatment result preferentially following crosslink formation. These results indicate that XPF is required to form gamma-H2AX and likely double strand breaks in response to interstrand crosslinks in human cells. Furthermore, XPA may be important to allow psoralen interstrand crosslinks to be processed without forming a double strand break intermediate.     

Survival of apurinic SV40 DNA in the d-complementation group of xeroderma pigmentosum.

The survival of depurinated Form I SV40 DNA was studied in normal human fibroblasts and in D-complementation Xeroderma pigmentosum (XP) fibroblasts. Survival was measured with an infective center assay. Heat-acid and methyl methanesulfonate (MMS) were used as depurinating agents. After 3 hrs of depurination by heat--acid treatment, infectivity in normal cells was less than 15% of the controls compared to more than 50% for the XP D cell strains. Similar results were obtained with MMS-treated DNA. These results are contrary to expectation since apurinic endonuclease activity, which is presumed to be involved in the repair of apurinic sites, is much lower in XP D cell strains than in normal cell strains. Our results indicate that another mechanism for the repair of apurinic sites could exist.     

A comparison of the response of unstimulated and stimulated T-lymphocytes and fibroblasts from normal, xeroderma pigmentosum and trichothiodystrophy donors to the lethal action of UV-C.

Unstimulated T-lymphocytes from normal donors are significantly more sensitive to the lethal effects of UV-C than either stimulated T-lymphocytes or fibroblasts as judged by colony-forming ability. Data from other studies suggest that excision repair is more effective in stimulated than unstimulated T-lymphocytes leading to the prediction that these differences in survival should be minimal in cells established from excision defective donors. The prediction was met with XP6BR, a donor of unknown complementation group. For 3 XP's from complementation group D, however, enhanced survival in stimulated T-cells was observed. With cells from an excision-defective TTD who was included in complementation group D of XP both fibroblasts and unstimulated T-lymphocytes were hypersensitive. For a second excision defective TTD patient who was excluded from complementation group D, the unstimulated T-lymphocytes were more resistant than those of normal donors although the fibroblasts were hypersensitive. These results suggest that the in vitro response of stimulated T-lymphocytes or fibroblasts may not reflect the in vivo response of cells, as measured by the response of unstimulated T-lymphocytes.     

The action of AF 2 on cultured hamster and human cells under aerobic and hypoxic conditions

AF 2 (2-(2-furyl)-3-(5-nitro-furyl)acrylamide) was toxic to Chinese hamster V 79 cells and normal human fibroblasts in aerobic media. However, the toxicity of the drug was increased many times by hypoxia. Similarly, the frequency of AF 2-induced azaguanine- and ouabain-resistant mutants of V 79 cells was much higher in hypoxia than under aerobic conditions. Both hamster V 79 cells and human fibroblasts metabolized AF 2 and other nitrofurans rapidly only under hypoxic conditions. Human fibroblasts were more sensitive to AF 2 both under aerobic conditions and in hypoxia than were V 79 cells under similar conditions. The Chinese hamster cells consistently gave survival curves with marked shoulders while human cells did not. Aerobic cultures of fibroblasts derived from xeroderma pigmentosum (XP) patients were markedly sensitive to AF 2 while fibroblasts from two ataxia telangeictasia patients had normal sensitivity. Under hypoxic conditions the sensitivity of both types of cells was increased but the XP line remained 5--10-fold more sensitive than normal or ataxia cells. These results suggest that the DNA lesions produced by AF 2 may be regarded as similar to those produced by ultraviolet light, at least in terms of their repairability in human cells.     

Transforming genes in human tumors

DNAs isolated from a variety of human tumor cell lines as well as from naturally occurring human carcinomas and sarcomas were shown to induce morphologic transformation upon transfection into NIH/3T3 cells. All tested transformants contained human DNA sequences, some of which specifically cosegregated with the malignant phenotype in additional cycles of transfection. Southern blot analysis of second cycle transformants derived from T24 human bladder carcinoma cells showed the presence of a single 15 kbp EcoRI fragment of human DNA. These sequences were molecularly cloned utilizing λ Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated λ T24-15A, was shown to contain an internal 6.6 kbp Bam HI fragment of human DNA that transformed NIH/3T3 fibroblasts with a specific activity of 5 × 10⁴ focus forming units per picomol. These results indicate that we have moleculary cloned an oncogene present in T24 bladder carcinoma cells. Comparison of molecular clones containing the T24 oncogene and its normal homologue did not reveal biochemical differences that helped to explain the malignant properties of this oncogene. Finally, we report preliminary results indicating that the T24 bladder carcimoma oncogene is highly related to the transforming gene of BALB-MSV, an acute transforming retrovirus.     

Ultraviolet mutational spectrum in a shuttle vector propagated in xeroderma pigmentosum lymphoblastoid cells and fibroblasts

In order to examine possible cell-type specificity in mutagenic events, a shuttle-vector plasmid, pZ189, carrying a bacterial suppressor tRNA marker gene, was treated with ultraviolet radiation and propagated in Epstein-Barr virus transformed lymphoblastoid cell lines from a patient, XP12BE, with xeroderma pigmentosum (XP), group A, and a normal control. XP is a skin-cancer-prone disorder with UV hypersensitivity and defective DNA repair. Plasmid survival and mutations inactivating the marker gene were scored by transforming an indicator strain of E. coli. An earlier report on this data [Seetharam et al., (1990) J. Mol. Biol., 212, 433] indicated lower survival and higher mutation frequency with the UV-treated plasmid passed through the XP12Be(EBV) line. In the present report, sequence analysis of 198 mutant plasmids revealed a predominance of G:C----A:T transitions with both lymphoblastoid cell lines. This finding is consistent with the bias of polymerases toward insertion of an adenine opposite non-coding photoproducts (dinucleotides or other lesions). Transversion mutagenesis, non-adjacent double mutations, and triple-base mutations may involve other mechanisms. These results were compared to similar data from a fibroblast line from the same patient [Bredberg et al., (1986) Proc. Natl. Acad. Sci. (U.S.A.), 83, 8273]. The frequency of G:C----A:T transitions was higher, and there were fewer plasmids with multiple-base substitutions and with transversion mutations with both XP lymphoblasts and fibroblasts than with the normal lymphoblasts and fibroblasts. There were no significant differences in classes or types of mutations in the UV-treated plasmid replicated in the XP lymphoblasts and the XP fibroblasts. This suggests that the major features of UV mutagenesis in different cell types from the same individual are similar.     

Survival and herpes virus production of normal and xeroderma pigmentosum fibroblasts after treatment with formaldehyde.

The survival of excision-deficient and of excision-proficient (variant) skin fibroblasts from xeroderma pigmentosum (XP) donors was about 5 times and twice, respectively, more sensitive to formaldehyde (FA) treatment than that of skin fibroblasts from healthy and XP heterozygote donors. The capacity of FA-treated host cells to further support Herpes virus (HSV) replication was also more sensitive to FA in XP12BE (group A) than in normal (KD) cells. An important recovery of this capacity occurred in both cell types when they were infected at increasing times (up to 36 h) after FA treatment. This contrasts with the decreasing capacity observed in XP12BE when similarly infected at increasing times after exposure to ultraviolet. In addition, the survival of FA-treated HSV was comparable in KD and XP12BE cells, whereas that of UV-irradiated HSV was much lower in XP12BE than in KD cells.