The frequency and accuracy of replication past a thymine-thymine cyclobutane dimer are very different in Saccharomyces cerevisiae and Escherichia coli.

We have compared the mutagenic properties of a T-T cyclobutane dimer in baker's yeast, Saccharomyces cerevisiae, with those in Escherichia coli by transforming each of these species with the same single-stranded shuttle vector carrying either the cis-syn or the trans-syn isomer of this UV photoproduct at a unique site. The mutagenic properties investigated were the frequency of replicational bypass of the photoproduct, the error rate of bypass, and the mutation spectrum. In SOS-induced E. coli, the cis-syn dimer was bypassed in approximately 16% of the vector molecules, and 7.6% of the bypass products had targeted mutations. In S. cerevisiae, however, bypass occurred in about 80% of these molecules, and the bypass was at least 19-fold more accurate (approximately 0.4% targeted mutations). Each of these yeast mutations was a single unique event, and none were like those in E. coli, suggesting that in fact the difference in error rate is much greater. Bypass of the trans-syn dimer occurred in about 17% of the vector molecules in both species, but with this isomer the error rate was higher in S. cerevisiae (21 to 36% targeted mutations) than in E. coli (13%). However, the spectra of mutations induced by the latter photoproduct were virtually identical in the two organisms. We conclude that bypass and error frequencies are determined both by the structure of the photoproduct-containing template and by the particular replication proteins concerned but that the types of mutations induced depend predominantly on the structure of the template. Unlike E. coli, bypass in S. cerevisiae did not require UV-induced functions.     

UVA-induced cyclobutane pyrimidine dimers form predominantly at thymine-thymine dipyrimidines and correlate with the mutation spectrum in rodent cells

Ligation-mediated PCR was employed to quantify cyclobutane pyrimidine dimer (CPD) formation at nucleotide resolution along exon 2 of the adenine phosphoribosyltransferase (aprt) locus in Chinese hamster ovary (CHO) cells following irradiation with either UVA (340–400 nm), UVB (295–320 nm), UVC (254 nm) or simulated sunlight (SSL; λ > 295 nm). The resulting DNA damage spectrum for each wavelength region was then aligned with the corresponding mutational spectrum generated previously in the same genetic target. The DNA sequence specificities of CPD formation induced by UVC, UVB or SSL were very similar, i.e., in each case the overall relative proportion of this photoproduct forming at TT, TC, CT and CC sites was ~28, ~26, ~16 and ~30%, respectively. Furthermore, a clear correspondence was noted between the precise locations of CPD damage hotspots, and of ‘UV signature’ mutational hotspots consisting primarily of C→T and CC→TT transitions within pyrimidine runs. However, following UVA exposure, in strong contrast to the above situation for UVC, UVB or SSL, CPDs were generated much more frequently at TT sites than at TC, CT or CC sites (57% versus 18, 11 and 14%, respectively). This CPD deposition pattern correlates well with the strikingly high proportion of mutations recovered opposite TT dipyrimidines in UVA- irradiated CHO cells. Our results directly implicate the CPD as a major promutagenic DNA photoproduct induced specifically by UVA in rodent cells.     

Reduced efficiency and increased mutagenicity of translesion DNA synthesis across a TT cyclobutane pyrimidine dimer, but not a TT 6-4 photoproduct, in human cells lacking DNA polymerase eta

Xeroderma pigmentosum variant (XPV) patients carry germ-line mutations in DNA polymerase eta (poleta), a major translesion DNA synthesis (TLS) polymerase, and exhibit severe sunlight sensitivity and high predisposition to skin cancer. Using a quantitative TLS assay system based on gapped plasmids we analyzed TLS across a site-specific TT CPD (thymine-thymine cyclobutane pyrimidine dimer) or TT 6-4 PP (thymine-thymine 6-4 photoproduct) in three pairs of poleta-proficient and deficient human cells. TLS across the TT CPD lesion was reduced by 2.6-4.4-fold in cells lacking poleta, and exhibited a strong 6-17-fold increase in mutation frequency at the TT CPD. All targeted mutations (74%) in poleta-deficient cells were opposite the 3'T of the CPD, however, a significant fraction (23%) were semi-targeted to the nearest nucleotides flanking the CPD. Deletions and insertions were observed at a low frequency, which increased in the absence of poleta, consistent with the formation of double strand breaks due to defective TLS. TLS across TT 6-4 PP was about twofold lower than across CPD, and was marginally reduced in poleta-deficient cells. TLS across TT 6-4 PP was highly mutagenic (27-63%), with multiple mutations types, and no significant difference between cells with or without poleta. Approximately 50% of the mutations formed were semi-targeted, of which 84-93% were due to the insertion of an A opposite the template G 5' to the 6-4 PP. These results, which are consistent with the UV hyper-mutability of XPV cells, highlight the critical role of poleta in error-free TLS across CPD in human cells, and suggest a potential involvement, although minor, of poleta in TLS across 6-4 PP under some conditions.     

Neutron radiation can activate K-ras via a point mutation in codon 146 and induces a different spectrum of ras mutations than does gamma radiation.

Neutron radiation is known to produce tumors in animals and cause cell transformation. We have developed a protocol to efficiently induce thymic lymphomas in RF/J mice by a single acute dose of neutron irradiation. Activated ras genes were detected in 17% (4 of 24) of the tumors analyzed. One of the tumors contained a K-ras gene activated by a point mutation in codon 146. Activating ras mutations at position 146 have not been previously detected in any known human or animal tumors. The spectrum of ras mutations detected in neutron radiation-induced thymic lymphomas was different from that seen in thymic lymphomas induced by gamma radiation in the same strain of mice. These results may have important implications for the mechanisms by which different types of radiation damage DNA.     

Identification of a single nucleotide C-to-T transition and five different deletions in patients with severe hemophilia B.

DNA of 70 unrelated hemophilia B patients, including three inhibitor patients, was analyzed by using various restriction enzymes and was hybridized with both a factor IX cDNA and 3'- and 5'-flanking probes. When the gene was mapped this way, six patients all afflicted with severe hemophilia B were shown to have a deviating hybridization pattern. One inhibitor patient showed a partial deletion of about 9 kb that removes exons a-c. A partial deletion of at least 11 kb that removed exon a and that had a maximum size of 35 kb in the 5'-flanking region could be identified in a patient of unknown status. In another three noninhibitor patients a complete deletion of the factor IX gene and two partial deletions could be observed. The partial deletions are of approximately 8 kb and approximately 1.5 kb, removing exons d and e and exon g, respectively. As detected by oligonucleotide probing, a C-to-T transition at amino acid 338 gave rise to an altered TaqI restriction pattern that could be observed in a sixth patient. The other 64 hemophilia B patients, including two inhibitor patients, showed a hybridization pattern indistinguishable from a normal one.     

Impaired nuclear functions lead to increased senescence and inefficient differentiation in human myoblasts with a dominant p.R545C mutation in the LMNA gene

We have studied myoblasts from a patient with a severe autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD) caused by an arginine 545 to cystein point mutation (p.R545C) in the carboxy-terminal domain of the lamin A/C gene. This mutation has pleiotropic cellular effects on these myoblasts as demonstrated by nuclear structural defects, exhibiting lobulations which increase with cell passages in culture. The organization of both lamin A/C and its inner nuclear membrane partner emerin are altered, eventually showing a honeycomb pattern upon immunofluorescence microscopy. In addition, the distribution of histone H3 trimethylated at lysine 27 and of phosphorylated RNA polymerase II, markers of inactive and active chromatin domains, respectively, are altered suggesting an impact on gene expression. Patient myoblasts also presented a high index of senescence in ex vivo culture. Moreover, our data show for the first time in an AD-EDMD context that the 20S core particle of the proteasome was inactivated. With cell passages, the 20S core protein progressively accumulated into discrete nuclear foci that largely colocalized with promyelocytic leukemia (PML) bodies while p21 accumulated throughout the nuclear compartment. Proteasome inactivation has been linked to normal cellular ageing. Our data indicate that it may also contribute to premature senescence in AD-EDMD patient myoblasts. Finally, when transferred to low-serum medium, patient myoblasts were deficient in ex vivo differentiation, as assessed by the absence of myotube formation and myogenin induction. Altogether, these data suggest that the LMNA mutation p.R545C impairs both proliferation and differentiation capacities of myoblasts as part of the pathogenesis of AD-EDMD.     

Human endothelial cells are activated by IFN-gamma to inhibit Toxoplasma gondii replication. Inhibition is due to a different mechanism from that existing in mouse macrophages and human fibroblasts

Toxoplasma gondii invaded and proliferated in cultured human umbilical vein endothelial cells. Preincubation of the human umbilical vein endothelial cells with human rIFN-gamma induced a high degree of inhibition of T. gondii replication, with the effect being dose dependent. In order to try to elucidate the inhibitory mechanism, we tested the presence of several factors that are known to operate against intracellular parasites in other cell types. We found, by means of a competitive inhibitor, that L-arginine-dependent production of reactive nitrogen intermediates was not the cause of inhibition of T. gondii proliferation, thus contrasting with the inhibitory mechanism found in activated mouse macrophages. Furthermore, the inhibition of replication was not overcome by oxygen scavengers or by saturation of the system with tryptophan, suggesting that neither reactive oxygen intermediates nor the induction of tryptophan starvation was responsible.     

Studies on 17-1A antigen gene regulation in nonexpressing A549 and A431 cells,as compared to expressing pancreatic carcinoma (CAPAN 2) cells,reveal a complex mechanism of repression of this gene.

Elements controlling high expression of the 17-1A antigen gene in pancreatic carcinoma cells (Capan 2) reside within the two regions: proximal (?193 to +3) and distal (?877 to ?518). We demonstrate here that some factors present in nuclear extracts from nonexpressing cells bind specifically to the control elements, important for gene expression. Our results suggest that nonexpressing cells may either lack at least one of the factors necessary for activation or may contain their modified forms. A major difference between expressing and nonexpressing cells was found in the region containing core enhancer sequence. Moreover, nonexpressing cells display a complex pattern of DNA-protein interactions in this region, suggesting that these cells contain factors acting negatively mainly on the enhancer sequence. Our results however, indicate that the mechanism of repression is much more complicated than expected.     

Rate of DNA synthesis in mammalian cells irradiated with ultraviolet light: a model based on the variations in the rate of movement of the replication fork and in the number of active replicons

A model is proposed to describe the rate of DNA synthesis observed under certain conditions in UV irradiated mammalian cells. It is assumed that shortly after irradiation the rate of DNA synthesis drops mainly as a consequence of the drop in the rate of movement of the replication fork. This in turn, is due to a pause at the dimer for a limited length of time. Later on, a recovery in the rate of DNA synthesis occurs, and it is proposed that one of the parameters contributing to that is an increase in the number of active replicons. This simple model enables one to predict variations in the rate of DNA synthesis as a function both of UV dose and of time after irradiation.     

Anti-CD3 and concanavalin A-induced human T cell proliferation is associated with an increased rate of arachidonate-phospholipid remodeling. Lack of involvement of group IV and group VI phospholipase A2 in remodeling and increased susceptibility of proliferating T cells to CoA-independent transacyclase inhibitor-induced apoptosis

In this study arachidonate-phospholipid remodeling was investigated in resting and proliferating human T lymphocytes. Lymphocytes induced to proliferate with either the mitogen concanavalin A or with anti-CD3 (OKT3) in combination with interleukin 2 (OKT3/IL-2) showed a greatly accelerated rate of [3H]arachidonate-phospholipid remodeling compared with resting lymphocytes or with lymphocytes stimulated with OKT3 or IL-2 alone. The concanavalin A-stimulated cells showed a 2-fold increase in the specific activity of CoA-independent transacylase compared with unstimulated cells, indicating that this enzyme is inducible. Stimulation with OKT3 resulted in greatly increased quantities of the group VI calcium-independent phospholipase A2 but not of the quantity of group IV cytosolic phospholipase A2. However, group IV phospholipase A2 became phosphorylated in OKT3-stimulated cells, as determined by decreased electrophoretic mobility. Incubation of cells with the group VI phospholipase A2 inhibitor, bromoenol lactone, or the dual group IV/group VI phospholipase A2 inhibitor, methyl arachidonyl fluorophosphonate, did not block arachidonate-phospholipid remodeling resting or proliferating T cells, suggesting that these phospholipases A2 were not involved in arachidonate-phospholipid remodeling. The incubation of nonproliferating human lymphocytes with inhibitors of CoA-independent transacylase had little impact on cell survival. In contrast, OKT3/IL-2-stimulated T lymphocytes were very sensitive to apoptosis induced by CoA-independent transacylase inhibitors. Altogether these results indicate that increased arachidonate-phospholipid remodeling is associated with T cell proliferation and that CoA-independent transacylase may be a novel therapeutic target for proliferative disorders.     

Photo-chemically induced DNA effects in the comet assay with epidermal cells of SKH-1 mice after a single oral administration of different fluoroquinolones and 8-methoxypsoralen in combination with exposure to UVA

Due to the need for in vivo photo-genotoxicity tests, the in vivo photo-comet assay was established in epidermal cells of the SKH-1 mouse. Groups of 10 male SKH-1 mice each were treated once orally with vehicle only, with three fluoroquinolones (25 mg/kg clinafloxacin, 20 mg/kg lomefloxacin, 200 mg/kg ciprofloxacin) or with 200mg/kg 8-methoxypsoralene (8-MOP). Thirty minutes after treatment half of the mice in each group were exposed to 23.8 J/cm2 UVA. Thereafter the mice were killed and their epidermal cells tested in the alkaline (pH >13) comet assay; at the same time after administration, compound-treated, non-irradiated mice were killed and analysed. A negative control group of ten male SKH-1 mice received the vehicle only; half of these animals were exposed to UVA, half were not. The comet tail lengths of epidermal cells of the mice were statistically significantly increased for all three fluoroquinolones (FQ) tested in combination with UV irradiation. Treatment with 8-methoxypsoralene+UV induced a significant reduction of comet tail length. Tail intensity and tail moment gave essentially the same results after combined exposure (compound+UV). Without irradiation, the tail lengths of controls and compound-treated mice were comparable under the conditions of this study. In contrast, tail intensity and tail moment were increased for all test compounds (including 8-MOP), without irradiation. Irradiated controls had a tail length comparable to non-irradiated controls, while tail intensity and tail moment were clearly increased in irradiated controls. In conclusion: under the present experimental conditions the in vivo photo-comet assay is able to detect photo-chemically induced DNA strand breaks as well as photo-chemically induced DNA cross-links.     

TGF-beta-induced G2/M delay in proliferating rabbit articular chondrocytes is associated with an enhancement of replication rate and a cAMP decrease: possible involvement of pertussis toxin-sensitive pathway.

This study was undertaken to gain more insight into the mechanism whereby TGF-beta influences the cell cycle progression of cultured rabbit articular chondrocytes. Using proliferating chondrocytes in fetal calf serum-containing medium, we have previously shown that TGF-beta induced a recruitment of cells at the end of the S phase (G2/M) observed 24 h after addition. The delayed cells may then be released, producing a proliferative effect at 48 h, provided a substantial amount of FCS (10%) is present in the medium. Otherwise, in low level of serum (2% FCS, for example), only inhibition of cell proliferation is observed. In chondrocytes synchronized in S phase by a thymidine block, we investigated here the time-course incorporation of [3H]-thymidine into DNA, the cell cycle traverse by flow cytofluorometric study of DNA content, the expression of PCNA (Proliferating Cell Nuclear Antigen), and cAMP levels. The data demonstrate that TGF-beta provoked a decrease of cAMP content (0.5-1 h) followed by an enhancement of the DNA synthesis rate (4 h) which was detectable through cytofluorometric analysis and [3H]-thymidine labeling and correlated with the PCNA expression. In contrast, addition of cAMP analogues to the cultures resulted in an inhibition of replication rate. We also showed that pertussis toxin produced a decrease of the DNA synthesis rate, in a transient manner and only in the presence of TGF-beta. All these results suggest that TGF-beta may accelerate the replication process of cyclized chondrocytes, making then accumulate at the G2/M boundary, via a mechanism that could involve the adenylate cyclase activity and a Gi-protein. The factor might be responsible for producing a pool of cells having already replicated their DNA and therefore capable of re-entering the cell cycle without delay. This cell population could serve as a tissue reserve able to induce a mitosis wave when necessary--for example, in the repair of tissue damage.     

Effects of metabolic inhibitors on cell lethality and mutation induction in Chinese hamster cells. I. Inhibitors of de novo purine synthesis and a comparison with the effects of caffeine

The effect of pre- and posttreatment incubation of UV-irradiated and ethyl methanesulphonate (EMS) treated cells with non-toxic concentrations of inhibitors of de novo purine synthesis (dnPS) on expression of potentially lethal and premutational damage at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in V79 cells has been examined. The concentrations of inhibitors used were shown to profoundly perturb de novo DNA synthesis, by measurements of [¹⁴C]formate uptake, and cell cycle progression by flow cytofluorimetry. Postincubation in 6-methyl mercaptopurine ribonucleoside (MMPR) usually but not invariably potentiated the cytotoxic effects of UV and EMS but azaserine (AZS) and methotrexate (MTX) were without effect. No effects on mutant frequencies were observed on posttreatment with any of these agents. Caffeine produced the least effect on dnPS, but invariably potentiated lethal damage. This potentiation of lethal damage is not mediated by dnPS inhibition as has been suggested for Chinese hamster ovary (CHO) cells.     

A single base mutation in type I procollagen (COL1A1) that converts glycine alpha 1-541 to aspartate in a lethal variant of osteogenesis imperfecta: detection of the mutation with a carbodiimide reaction of DNA heteroduplexes and direct sequencing of products of the PCR.

Skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta synthesized both apparently normal type I procollagen and a type I procollagen that had slow electrophoretic mobility because of posttranslational overmodifications. The thermal unfolding of the collagen molecules as assayed by protease digestion was about 2 degrees C lower than normal. It is surprising, however, that collagenase A and B fragments showed an essentially normal melting profile. Assay of cDNA heteroduplexes with a new technique involving carbodiimide modification indicated a mutation at about the codon for amino acid 550 of the alpha 1(I) chain. Subsequent amplification of the cDNA by the PCR and nucleotide sequencing revealed a single-base mutation that substituted an aspartate codon for glycine at position alpha 1-541 in the COL1A1 gene. The results here confirm previous indications that the effects of glycine substitutions in type I procollagen are highly position specific. They also demonstrate that a recently described technique for detecting single-base differences by carbodiimide modification of DNA heteroduplexes can be effectively employed to locate mutations in large genes.     

Dendritic cells recruitment and in vivo priming of CD8+ CTL induced by a single topical or transepithelial immunization via the buccal mucosa with measles virus nucleoprotein.

The buccal mucosa, a prototype of pluristratified mucosal epithelia, contains a network of directly accessible class II(+) epithelial dendritic cells (DC), similar to skin Langerhans cells. We showed that a single buccal immunization with measles virus nucleoprotein (NP), by either topical application onto or intradermal injection in the buccal mucosa, induced in vivo priming of protective class I-restricted specific CD8(+) CTL. Both routes of immunization with NP induced a rapid recruitment of DC into the mucosa, which peaked at 2 h and decreased by 24 h. Treatment of mice with Flt3 ligand resulted in an increased number of DC in the buccal mucosa and enhanced the frequency of IFN-gamma-producing NP-specific effectors and the NP-specific CTL response generated after buccal immunization with NP. Finally, NP-pulsed bone marrow-derived DC induced NP-specific IFN-gamma-producing cells upon adoptive transfer to naive mice. These data demonstrate that a viral protein delivered to DC of the buccal mucosa induces in vivo priming of protective anti-viral CD8(+) CTL.     

Chalcone isomerase isozymes with different substrate specificities towards 6'-hydroxy- and 6'-deoxychalcones in cultured cells of Glycyrrhiza echinata, a leguminous plant producing 5-deoxyflavonoids.

Three chalcone isomerase isozymes in Glycyrrhiza echinata (Fabaceae) were separated by chromatofocusing and partially purified to examine their substrate specificities. Two isozymes, one of which was elicitor-inducible, acted on both 6'-hydroxychalcone and 6'-deoxychalcone and presumably are involved in the legume-specific 5-deoxyflavonoid pathway, while another acted on only 6'-hydroxychalcone.     

Mutagenicity testing on chinese hamster V79 cells treated in the in vitro liver perfusion system. Comparative investigation of different in vitro metabolising systems with dimethylnitrosamine and benzo[a]pyrene.

A comparative study of three in vitro metabolising systems was performed in combination with Chinese hamster V79 cells, at which point mutation to 6-thioguanine resistance was scored. The three metabolising systems used were: (1) rat liver microsomal fraction (S9-mix); (2) feeder layer of primary embryonic golden hamster cells, according to Hubermann's system; (3) in vitro perfusion of rat liver according to the system of Beije et al. As model substances dimethylnitrosamine (DMN) and benzo[a]pyrene (BP) was used. The liver perfusion was more efficient than S9-mix as an activating system of DMN, while the feeder layer of embryonic cells was unable to activate this compound. The activation of DMN with S9-mix was dependent on the presence of NADP. By exposing the target cells in the liver perfusion at different distances from the liver the biological half life of the active metabolite of DMN could be estimated to less than 5 s. With BP the three metabolising systems showed reversed results as compared with DMN--both the feeder layer cells and S9-mix activated BP, the feeder layer cells being most efficient. With liver perfusion, the perfusate itself was totally negative. Only the bile showed a week mutagenic effect. These results are in accordance with the notion that intact liver cells perform both an activation and a subsequent deactivation of BP. Because of the importance of hepatic bio-transformation in chemical mutagenesis and carcinogenesis it is emphasied that a liver perfusion system could be used in a testing protocol for genotoxic effects as a valuable tool in order to analyse the mechanism of action of mutagenic and carcinogenic compounds detected in other test systems, for instance bacterial/microsomal tests.