The role of CSA in the response to oxidative DNA damage in human cells.

Cockayne syndrome (CS) is a rare genetic disease characterized by severe growth, mental retardation and pronounced cachexia. CS is most frequently due to mutations in either of two genes, CSB and CSA. Evidence for a role of CSB protein in the repair of oxidative DNA damage has been provided recently. Here, we show that CSA is also involved in the response to oxidative stress. CS-A human primary fibroblasts and keratinocytes showed hypersensitivity to potassium bromate, a specific inducer of oxidative damage. This was associated with inefficient repair of oxidatively induced DNA lesions, namely 8-hydroxyguanine (8-OH-Gua) and (5'S)-8,5'-cyclo 2'-deoxyadenosine. Expression of the wild-type CSA in the CS-A cell line CS3BE significantly decreased the steady-state level of 8-OH-Gua and increased its repair rate following oxidant treatment. CS-A cell extracts showed normal 8-OH-Gua cleavage activity in an in vitro assay, whereas CS-B cell extracts were confirmed to be defective. Our data provide the first in vivo evidence that CSA protein contributes to prevent accumulation of various oxidized DNA bases and underline specific functions of CSB not shared with CSA. These findings support the hypothesis that defective repair of oxidative DNA damage is involved in the clinical features of CS patients.

Tumor suppression by DNA base excision repair.

Base excision repair (BER) is the main pathway for repair of endogenous DNA damage. It was expected that different tumor types could derive from BER defects but to date this link is elusive. In vitro and molecular epidemiology studies may be used to unravel this issue.

Reduced ligation during DNA base excision repair supported by BRCA2 mutant cells.

The breast cancer predisposing genes BRCA1 and BRCA2 appear to be involved in DNA repair. In particular, the sensitivity of BRCA2-deficient mouse embryonic fibroblasts to ionizing radiation and the demonstrated interaction of the BRCA2 protein with Rad51, a major factor in recombinational repair, indicate that BRCA2 is important for double strand break repair. The human BRCA2-deficient human cell line Capan-1, whilst being sensitive to ionizing radiation, is also sensitive to the alkylating agent methymethanesulfonate. The major lesions induced by this agent are methylated bases which are removed primarily by the base excision repair (BER) pathway. We have investigated the efficiency of BER in Capan-1 cells by an in vitro assay in which plasmid substrates containing a single lesion are repaired by mammalian cell extracts. In comparison to the control cell lines BxPC-3, T24 and MCF7, Capan-1 cells exhibited a reduced rate of DNA ligation during both the single-nucleotide insertion and PCNA-dependent pathways of BER. The reduced rate of DNA ligation exhibited by Capan-1 cell extracts was complemented by addition of bacteriophage T4 DNA ligase or human DNA ligase III. BRCA2-mutant Capan-1 cells may possess reduced DNA ligase activity during BER.

O6-alkylguanine-DNA alkyltransferase activity in human brain tumors.

The O6-alkylguanine-DNA alkyltransferase (AT) activity in different kinds of human brain tumors was investigated. Twenty-seven brain tumors were analysed. Twenty-five of them showed proficient AT activity with values ranging between 20 and 722 fmol AT/mg protein. The two AT-deficient tumors observed were an oligodendroglioma and an astrocytoma. The relationship between the different histological kinds of tumor, with respect to the AT activity was: meningeomas greater than sarcomas greater than glioblastomas greater than astrocytomas greater than oligodendrogliomas greater than neurinomas greater than lymphomas. The proposal of Kohn (DNA filter elution methods in anticancer drug development. In: Concepts, Clinical Developments, and Therapeutic Advances in Cancer Chemotherapy. Editor: F.M. Muggia. Martinus Nijhoff Publishers, Boston) to confine treatments with alkylating antineoplastic agents to AT-deficient tumors, is discussed.

Age-independency of AP site incision capacity in women.

Women in the age range 40-59 years have a >4 fold higher risk to develop cancer as compared to women in the age range birth-39 years. This age-related increase in cancer incidence might be partially linked to reduced efficiency of the DNA repair machinery. We have investigated the abasic (AP) site incision capacity of peripheral blood lymphocytes (PBL) from 23 women in the age range 27-57 years. The AP sites incision capacity was determined in relation to protein or DNA content of PBL extracts. In either case, no significant correlation was found between AP sites incision capacity and age, thus suggesting that no decline occurs in the age range investigated.

Radiation treatment plus CCNU plus the radiosensitizer lonidamine in malignant gliomas operated.

From June 1988 to January 1990, 28 patients with primary brain tumours were operated and treated with radiotherapy (RT) (50 Gy whole brain + 10 Gy boost to tumour bed) + cyclohexylnitrosourea (CCNU) 130 mg/msq p.o. every 6 weeks + the radiosensitizer Lonidamine (LND) (150 mg T.I.D. for the whole duration of treatment). Myelotoxicity of this regimen was acceptable, with two cases of grade IV leukopenia and thrombocytopenia requiring discontinuation of treatment. LND was discontinued in 6 patients for major toxicity (myalgias and/or testicular pain), and 3 additional patients required dose reduction of this drug. The median follow-up time of the patients on study was 12 months. The median survival time (MST) was 5 months for grade IV astrocytomas (n = 8) and 16 months for grade III lesions (= 20). No correlation was seen between survival of patients and DNA content, measured by flow cytometry, or levels of O6- alkylguanine-DNA alkyltransferase, an enzyme that repairs the CCNU-induced DNA damage.

Influence of RNA synthesis on DNA-repair replication in human cell extracts.

An in vitro system allowing human cell extracts to carry out excision repair in ultraviolet light-irradiated, closed circular DNA was used to study the influence of transcription on repair synthesis. RNA synthesis from plasmid DNA containing bacterial transcription units was obtained by addition of Escherichia coli RNA polymerase and ribonucleoside triphosphates to the repair-incubation mixture. No increase in UV-stimulated repair replication was observed under transcriptional conditions; in fact, an inhibition of repair replication occurred, possibly due to impairment of DNA polymerization. This is unlike the in vivo situation where preferential DNA repair of transcribed genes has been found after UV-irradiation. Possible factors important for preferential repair of expressed genes in mammalian cells are discussed.

Negative evidence for an adaptive response to lethal and mutagenic effects of alkylating agents in V79 Chinese hamster cells.

V79 Chinese hamster cells were subjected to a number of different pretreatment regimens with low concentrations of N-methyl-N-nitrosourea (MNU), N-ethyl-N-nitrosourea (ENU) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). They were then treated with either MNU or ENU at toxic and mutagenic concentrations. No differences in cell killing or mutation induction to 6-thioguanine resistance were found between pretreated and non-pretreated cells. These results argue against the existence of an adaptive response to alkylating agents in this cell line.

Stem cell-mediated delivery of therapies in the treatment of glioma.

High grade gliomas can be seldom controlled, due to the infiltrative nature of these tumors and the presence of cell populations resistant to radio- and chemotherapy. Current research aims to develop novel therapeutic approaches to track and eliminate the disseminated glioma-driving cells. Selected delivery of therapeutic agents taking advantage of the tropism of normal stem cells for glioma cells might be one.

Increased resistance to oxidative DNA damage of trabecular meshwork cells by E. coli FPG gene transfection.

Oxidative damage plays a pathogenic role in various chronic degenerative diseases. Oxidative damage targeting trabecular meshwork (TM) cells as a consequence of mitochondrial damage is a pathogenic mechanism for glaucoma, the most common cause of irreversible blindness worldwide. Consequences of oxidative damage are attenuated by endocellular activities involved in scavenging reactive oxidative species and DNA repair. Selected bacterial genes are highly efficient at protecting cells from oxidative DNA damage. This situation occurs for Escherichia coli formamidopyrimidine DNA glycosylase (FPG), a major DNA glycosylase that repairs oxidatively damaged DNA. Accordingly, this study was aimed at transfecting human TM cells (HTMC) with Fpg in order to increase their resistance to oxidative damage. This study demonstrates that it is feasible to increase resistance of HTMC to endogenous oxidative damage by gene transfection. These findings bear relevance for primary and secondary prevention of degenerative glaucomas and other degenerative diseases where oxidative damage plays a pathogenic role.

Overexpression of enzymes that repair endogenous damage to DNA.

A significant contribution to human mutagenesis and carcinogenesis may come from DNA damage of endogenous, rather than exogenous, origin. Efficient repair mechanisms have evolved to cope with this. The main repair pathway involved in repair of endogenous damage is DNA base excision repair. In addition, an important contribution is given by O6-alkylguanine DNA alkyltranferase, that repairs specifically the miscoding base O6-alkylguanine. In recent years, several attempts have been carried out to enhance the efficiency of repair of endogenous damage by overexpressing in mammalian cells single enzymatic activities. In some cases (e.g. O6-alkylguanine DNA alkyltransferase or yeast AP endonuclease) this approach has been successful in improving cellular protection from endogenous and exogenous mutagens, while overexpression of other enzymatic activities (e.g. alkyl N-purine glycosylase or DNA polymerase beta) were detrimental and even produced a genome instability phenotype. The reasons for these different outcomes are analyzed and alternative enzymatic activities whose overexpression may improve the efficiency of repair of endogenous damage in human cells are proposed.

Counteracting spontaneous transformation via overexpression of rate-limiting DNA base excision repair enzymes.

DNA damage of endogenous origin may significantly contribute to human cancer. A major pathway involved in DNA repair of endogenous damage is DNA base excision repair (BER). BER is rather efficient in human cells but a certain amount of endogenous damage inevitably escapes mending and likely contributes to human carcinogenesis. Apart from some glycosylases that are particularly sluggish (e.g. 8-oxoG DNA glycosylase), recent work suggests that the general rate-limiting steps of BER may be trimming of 2-deoxyribose 5-phosphate in case the process is started by a monofunctional glycosylase or trimming of a 3'-blocking fragment, in case BER is started by a bifunctional glycosylase or in the case of single-strand breaks produced by free radical attack. Overexpression of the 5'-deoxyribophosphodiesterase (dRPase) domain of DNA polymerase beta, on the one hand, and of yeast APN1 protein, containing an efficient 3' repair activity, on the other, may lead to improved BER in mammals. The recently characterized S3 protein of Drosophila, containing both dRPase and 3'-trimming activities, could also be considered for overexpression studies. The possible protecting role of enhanced BER could be investigated in cultured rodent embryonic fibroblasts undergoing spontaneous transformation, a most interesting system that merits rediscovery.

The O6-methylguanine-DNA-methyltransferase activity of rat hepatoma cells is increased after a single exposure to alkylating agents.

The O6-methylguanine-DNA-methyltransferase activity was measured in rat hepatoma cells (H4 cells) at different times after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), methylmethane sulfonate (MMS) or ethylmethane sulfonate (EMS) treatment. Incubation with MNNG (10 microM) first depletes the methyltransferase activity, then the number of methyltransferase molecules per cell increases and reaches approximately 3-fold the constitutive level after 48 h. Incubations with MMS (0.5 or 1 mM) or with EMS (5 or 10 mM) do not modify or partially decrease the constitutive methyltransferase level. However, an enhancement of the activity is also observed after 48 h: the activity in 5- and 4-fold higher than the control value in MMS- and EMS-treated cells, respectively. The methyltransferase increase is due to de novo protein synthesis. It is not observed in cells constitutively lacking this protein. The data suggest that the O6-methylguanine (O6-meGua) repair capacity of H4 cells can be increased after a single treatment with alkylating agents, by a process different to the adaptive response.

Effect of topoisomerase poisoning by antitumor drugs VM 26, fostriecin and camptothecin on DNA repair replication by mammalian cell extracts.

A recently developed in vitro excision-repair system was used to investigate the effect of the topoisomerase poisons VM 26, fostriecin and camptothecin on DNA repair replication carried out by Chinese hamster ovary cell extracts. VM 26 and camptothecin partially inhibit topoisomerases II and I respectively, which are present in the repair-competent extracts, but have only slight effects on the repair efficiency. On the contrary, the antitumor drug fostriecin markedly affects repair replication but, in contrast to a previous report, does not seem to have, under the experimental conditions used, any inhibitory effect on topoisomerase II. This lack of correlation between the ability to inhibit DNA topoisomerases and the effect on DNA repair replication suggests that topoisomerases should not play a primary role in mammalian excision repair. The use of cleavable-complex stabilizing poisons to investigate the role of eukaryotic topoisomerases in DNA excision repair is discussed.

Comparative repair of the endogenous lesions 8-oxo-7, 8-dihydroguanine (8-oxoG), uracil and abasic site by mammalian cell extracts: 8-oxoG is poorly repaired by human cell extracts.

The repair of the endogenous lesions 8-oxo-7,8-dihydroguanine (8-oxoG), uracil (U) and natural abasic site (AP site) was investigated using an in vitro base excision repair assay in which a plasmid substrate containing a single lesion at a defined position was repaired by mammalian cell extracts. Repair replication of an 8-oxoG/cytosine base pair performed by normal human cell extracts was approximately 5-fold less efficient than repair of a U/adenine base pair and, in turn, the latter was repaired approximately 10-fold less efficiently than an AP site placed in front of an adenine. A similar pattern of repair capacity for the three lesions was observed in Chinese hamster extracts. Repair of 8-oxoG was performed by the one nucleotide insertion pathway only. The lower repair replication ability of 8-oxoG with respect to U was linked to a lower DNA glycosylase (base removal) activity rather than to inability to process the beta-elimination cleaved strand left by the AP lyase activity associated with human oxoguanine DNA glycosylase 1. The data show that DNA repair of 8-oxoG is poor in human cells in comparison with other frequent endogenous lesions.

Isolation and preliminary characterization of u.v.-sensitive mutants from the human cell line EUE.

Five u.v. light-sensitive clones were isolated in the EUE cell line by means of a modified form of the original 5-bromodeoxyuridine (BUdR)-light method worked out by Puck and Kao for the isolation of nutritional mutants. A cell population was mutagenized with ethylmethanesulfonate. After the expression time, cells were u.v.-irradiated and incubated with BUdR to label excision patches in repair proficient cells. A subsequent irradiation with 'black' light caused DNA strand breakage in BUdR-substituted cells. During BUdR treatment, hydroxyurea and a fluorochrome (Hoechst 33258) were added to possibly enhance the analogue incorporation into DNA and to increase the photolability of BUdR containing sequences, respectively. Out of 192 colonies selected with this method, 38 were isolated and tested for their u.v.-sensitivity. Five of them showed significant, reproducible differences with respect to the parental line. As a partial characterization, the five u.v.-sensitive clones were assayed for unscheduled [3H]thymidine incorporation after exposure to u.v. light, by means of liquid scintillation spectrometry and autoradiography. In all clones. DNA repair synthesis was significantly decreased with respect to the parental line.

Efficient DNA base excision repair in ataxia telangiectasia cells.

Ataxia telangiectasia (A-T) cells are sensitive to a broad range of free-radical-producing and alkylating agents. Damage caused by such agents is in part repaired by base excision [base excision repair (BER)]. Two BER pathways have been demonstrated in mammalian cells: a single-nucleotide-insertion pathway and a long-patch pathway involving resynthesis of 2-10 nucleotides. Although early studies failed to detect DNA-repair defects in A-T cells exposed to ionizing radiation and radiomimetic agents, more recent experiments performed in non-dividing A-T cells and the demonstrated interaction of the A-T-mutated protein (ATM) with the BRCA1 gene product suggest that a DNA-repair defect may underlie, at least in part, the radiation sensitivity in A-T cells. We have analysed BER of a single abasic site or a single uracil in two A-T families, using an in vitro BER system. In both families, the mutation involved was homozygous and completely inactivated the ATM protein. No difference was observed between affected individuals and heterozygous or homozygous wild-type relatives in their capacity to perform DNA repair by either one-nucleotide insertion or the long-patch pathway. Hence, the putative DNA-repair defect in A-T cells, if any, does not involve BER.

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.