Effect of UV light on DNA replication and chain elongation in Chinese hamster UV61 cells

Exposure of eukaryotic cells to ultraviolet light results in a temporary inhibition of DNA replication as well as a temporary blockage of DNA fork progression. Recently there has been considerable debate as to whether the (5-6)cyclobutane pyrimidine dimer, the pyrimidine(6-4)pyrimidone lesion or both are responsible for these effects. Using cell lines that repair both of these lesions (CHO AA8), only (6-4) lesions (CHO UV61) or neither (CHO UV5), we have shown that in rodent cells both lesions appear to play a role in both the inhibition of thymidine incorporation and the blockage of DNA fork progression. Specifically, after exposure to 2.5 J/m2, AA8 cells recover normal rates of DNA replication within 5 h after exposure, while UV5 cells exhibit a greater depression in thymidine incorporation for at least 10 h. UV61 cells, on the other hand, show an intermediate response, both with respect to the extent of the initial depression and the rate of recovery of thymidine incorporation. UV61 cells also exhibit an intermediate response with respect to blockage of DNA fork progression. In previous publications we have shown that UV5 cells exhibit extensive blockage of DNA fork progression and only limited recovery of this effect within the first 5 h after exposure to UV. In this report we show that UV61 cells exhibit a more extensive blockage of fork progression than is observed in AA8 cells. These blocks also appear to be removed (or overcome) more slowly than in the AA8 cells, but more rapidly than in UV5 cells. Taken together we conclude that both lesions appear to be involved in the initial depression in thymidine incorporation and the initial blockage of DNA fork progression in rodent cells. These data also indicate that (6-4) lesions may be responsible for the prolonged depression in thymidine incorporation and the prolonged blockage of DNA fork progression observed in UV5 cells.     

Involvement of nucleotide excision repair (NER) system in repair of mono ADP-ribosylated dG adducts produced by pierisin-1, a cytotoxic protein from cabbage butterfly

Pierisin-1, a cytotoxic protein from the cabbage butterfly (Pieris rapae), induces apoptosis in mammalian cell lines. Binding of its C-terminal region to glycosphingolipid Gb3 and Gb4 receptors on cell membrane is necessary for incorporation into cells, while the N-terminal polypeptide catalyzes transfer of the ADP-ribose moiety of NAD at N2 of dG in DNA. Resulting DNA adducts cause mutation if they are present at low levels. If the DNA damage is more severe, the cells undergo apoptosis. In the present study, we examined the repair system for ADP-ribosylated dG adducts using nucleotide excision repair (NER) mutants of Chinese hamster ovary (CHO) cell lines. Pierisin-1 showed cytotoxic effects in all cases: IC50 values of them were; 650 ng/ml for AA8 (wild), 230 ng/ml for UV5, 190 ng/ml for UV20, 260 ng/ml for UV41, and 240 ng/ml for UV135. Thus, wild-type AA8 proved most resistant to pierisin-1-induced cytotoxicity. When these CHO cell lines were treated with pierisin-1, the adduct levels of ADP-ribosylated dG increased to 2.5-4.8/10(5) nucleotides time-dependently in all cell lines at 12 h. After removal of pierisin-1, the adduct levels remained constant or increased to 4-14/10(5) nucleotides in all NER mutant cells (UV5, UV20, UV41, UV135), while those rapidly decreased to 0.27/10(5) nucleotides in the repair proficient AA8 cells for 24 h. From these results, it is suggested that the NER system is involved in the repair of ADP-ribosylated dG adducts in DNA.     

DNA replication in isolated nuclei : The fate of pulse-labelled DNA subunits

The fate of ³H-dTTP incorporated into DNA in isolated S phase nuclei from Chinese Hamster Ovary cells was examined. ³H-dTTP observed in 4 S DNA subunits after a pulse-label becomes acid-soluble during a chase performed under conditions which permit continued active DNA synthesis. ³H-dTTP incorporated into longer DNA subunits is not affected by these chase conditions. This selective loss of 4 S pulse-label requires active DNA synthesis. In incubations which do not permit continued DNA synthesis, either there is little loss of label or the loss occurs equally from the 4 S and larger DNAs. Possible reasons for a metabolically active 4 S subunit are discussed.     

Increasing Activity of Germinating Bacillus subtilis Spores to Incorporate Thymidine Triphosphate into Deoxyribonucleic Acid After Detergent Treatment

The incorporation of (3)H-labeled thymidine triphosphate ((3)H-dTTP) into deoxyribonucleic acid (DNA) of germinated and then Brij 58-treated Bacillus subtilis spores was measured to study DNA replication activity of cells. The dTTP incorporation rate was very low in dormant spores, gradually increased as germination proceeded, and reached a level of the vegetative cell activity approximately 4 hr after the start of germination. This is in contrast to the DNA polymerase activity in the cell extract which remained at the same level throughout the germination period. The increase of the dTTP incorporation activity was inhibited by chloramphenicol or phenethyl alcohol. When these inhibitors were added after germination had proceeded, the elevated dTTP incorporation activity gradually decreased. Permeability to dTTP of spores germinated in the presence of chloramphenicol and then treated with Brij 58 was confirmed by (i) (3)H-dTTP incorporation into the treated spores following either electron or ultraviolet irradiation and (ii) release of radioactivity from the treated spores containing radioactively labeled DNA after deoxyribonuclease I treatment.     

Effect of UV light on sister-chromatid exchanges, activation of alternative sites of replicon initiation and thymidine incorporation in CHO AA8, UV61 and UV5 cells.

The relative importance of the UV-induced pyrimidine(5-6)pyrimidine and the pyrimidine(6-4)-pyrimidone lesions in sister-chromatid exchanges (SCEs), activation of alternative sites of replicon initiation and thymidine incorporation were examined using wild-type Chinese hamster ovary (CHO) AA8 cells which remove both lesions, mutant CHO UV61 cells which remove only the (6-4) lesion and mutant CHO UV5 cells which remove neither lesion. Our data suggest that both lesions play a role in each end point examined. The relative importance of these lesions is dependent on the end point studied as well as the fluence used. For SCE induction and the activation of alternative sites of replicon initiation, the (6-4) lesion appears to play a predominant role, while for the thymidine incorporation studies the (6-4) lesion appears to play the predominant role at low fluences while the role of the (5-6) lesion increases at higher fluences.     

The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells

The majority of the high (12-fold elevated) baseline sister-chromatid exchanges (SCEs) that occur in the CHO mutant line EM9 appear to be a consequence of incorporated BrdUrd, and they arise during replication of DNA containing BrdUrd in a template strand. In normal CHO cells the alkaline elution patterns of DNA newly replicated on a BrdUrd-containing template are significantly altered compared with those seen during the replication on an unsubstituted template. The nascent DNA synthesized on such an altered template is delayed in reaching mature size, possibly because replication forks are temporarily blocked at sites occurring randomly along the template. Transient blockage of replication forks may be a prerequisite for SCE. The delay in replication on BrdUrd-substituted templates was greater in EM9 cells than in parental AA8 cells and was also greater in AA8 cells treated with benzamide, an inhibitor of poly(ADPR) polymerase, than in untreated AA8 cells. Under these conditions, treatment with benzamide also produced a 7-fold increase in SCEs in AA8. An EM9-derived revertant line that has a low baseline SCE frequency showed less delay in replication on BrdUrd-substituted templates than did EM9. However, under conditions where the template strand contained CldUrd, which was shown to produce 4-fold more SCEs than BrdUrd in AA8 cells, the replication delay in AA8 was not any greater in the CldUrd-substituted cells. Thus, other factors besides the delay appear to be involved in the production of SCEs by the template lesions resulting from incorporation of the halogen-substituted pyrimidine molecules.     

UV mutagenesis, cytotoxicity and split-dose recovery in a human-CHO cell hybrid having intermediate (6-4) photoproduct repair

Somatic cell hybrids constructed between UV-hypersensitive Chinese hamster ovary cell line UV20 and human lymphocytes were used to examine the influence of a human DNA repair gene, ERCC1, on UV photoproduct repair, mutability at several drug-resistance loci, UV cytotoxicity and UV split-dose recovery. In hybrid cell line 20HL21-4, which contains human chromosome 19, UV-induced mutagenesis at the APRT, HPRT and Na+/K+-ATPase loci was comparable to that in repair-proficient CHO AA8 cells, whereas cell line 20HL21-7, a reduced human-CHO hybrid not containing human chromosome 19, exhibited a hypermutable phenotype at all 3 loci indistinguishable from that of UV20 cells. The response of 20HL21-4 cells to UV cytotoxicity reflected substantial but incomplete restoration of wild-type UV cytotoxic response, whereas responses of UV20 and 20HL21-7 cell lines to UV cytotoxicity were essentially the same, reflecting several-fold UV hypersensitivity. Repair of UV-induced (5-6) cyclobutane dimers and (6-4) photoproducts was examined by radioimmunoassay; (6-4) photoproduct repair was deficient in UV20 and 20HL21-7 cell lines, and intermediate in 20HL21-4 cells relative to wild-type CHO AA8 cells. UV split-dose recovery in 20HL21-4 cells was also intermediate relative to AA8 cells. These results show that the human ERCC1 gene on chromosome 19 is responsible for substantial restoration of UV survival and mutation responses in repair-deficient UV20 cells, but only partially restores (6-4) UV photoproduct repair and UV split-dose recovery.     

Effect of ultraviolet light on thymidine incorporation, DNA chain elongation and replicon initiation in wild-type and excision-deficient Chinese hamster ovary cells

Wild-type Chinese hamster ovary cells (AA8) and five excision-deficient clones derived from the AA8 line (UV-4, UV-5, UV-20, UV-24 and UV-41) were exposed to ultraviolet light and then analyzed for their ability to incorporate [3H]thymidine and to initiate as well as elongate replicon-sized DNA fragments. After exposure to ultraviolet light, all cell lines exhibited a depression in the rate of thymidine incorporation. For exposures of 4.0 J/m2 or higher the wild-type cells recovered normal rates of thymidine incorporation within a few hours, while none of the excision-deficient lines exhibited complete recovery. For fluences below 4.0 J/m2 all but the UV-5 line exhibited at least some recovery. The ability to elongate DNA chains appeared to correlate with the thymidine incorporation data, with the UV-5 line exhibiting the strongest blockage of DNA chain elongation, the AA8 line exhibiting the least blockage, and the UV-20 line exhibiting an intermediate response. All cell lines exhibited a decrease in the distance between replication origins, thus supporting models which propose that exposure to ultraviolet light results in the use of alternative sites for the initiation of replication.     

Inhibition of transient gene expression in Chinese hamster ovary cells by cyclobutane dimers and (6-4) photoproducts in transfected ultraviolet-irradiated plasmid DNA

Using a transient gene expression assay to measure host cell reactivation, the effects of cyclobutane dimer and noncyclobutane dimer uv photoproducts on expression of a reporter gene were examined in normal and repair-deficient Chinese hamster ovary (CHO) cell lines. Ultraviolet damage in plasmid pRSV beta gal DNA, containing the Escherichia coli beta-galactosidase gene, resulted in reduced reporter gene expression in both uv-hypersensitive mutant CHO cell lines UV5 and UV61 relative to wild-type, parental AA8 cells. However, the effects of uv irradiation of transfected plasmid DNA on gene activity were reduced in UV61, a mutant with normal (6-4) photoproduct repair, compared to UV5, which is deficient in (6-4) photoproduct repair; this reduction correlated with the intermediate uv-hypersensitivity of UV61. Selective removal of cyclobutane dimers by in vitro photoreactivation of uv-irradiated plasmid DNA prior to transfection substantially increased reporter gene activity in both uv-hypersensitive mutant cell lines. This increase was significantly greater in UV61 than in UV5, consistent with UV5 being deficient in repair of both (6-4) photoproducts and cyclobutane dimers. These results suggest that unrepaired (6-4) photoproducts in transfected pRSV beta gal plasmid DNA are responsible for a significant fraction of the reduction in transient gene expression observed in recipient uv-hypersensitive CHO cell mutants.     

Increased uptake of thymidine in the activation of sea urchin eggs. III. Effects of aphidicolin

Uptake and phosphorylation of externally supplied [3H]-thymidine are fully stimulated in fertilized sea urchin eggs exposed to 5.0 micrograms/ml aphidicolin. As in untreated controls, the rate of uptake in aphidicolin-treated eggs increases greater than 50-fold shortly after fertilization, and greater than 85% of the transported thymidine is immediately phosphorylated to the triphosphate. The intracellular levels of [3H]-thymidine triphosphate (3H-dTTP) resulting from an external supply of [3H]-thymidine is therefore equal in aphidicolin-treated and untreated fertilized eggs. Under the same experimental conditions, the incorporation of externally supplied [3H]-thymidine into newly synthesized DNA of fertilized eggs is 90% inhibited by exposure to aphidicolin. The full availability of 3H-dTTP in these eggs further suggests that aphidicolin inhibits specifically at the level of DNA synthesis. This inhibitory effect is proportional to the concentration of aphidicolin between 0 and 5.0 micrograms/ml. In the continuous presence of 5.0 micrograms/ml aphidicolin, fertilized eggs fail to undergo mitotic chromosome condensation, nuclear envelope breakdown, and cytokinesis, suggesting a dependent link between these processes and the completion of nuclear DNA synthesis.     

DNA-ligase activities appear normal in the CHO mutant EM9

The Chinese hamster ovary (CHO) mutant strain EM9 was previously shown to be hypersensitive to killing by ethyl methanesulfonate (EMS) and methyl methanesulfonate (MMS), to have a 12-fold increased baseline incidence of sister-chromatid exchanges (SCE), and to be defective in rejoining DNA strand breaks after treatment with EMS, MMS, or X-rays. A study was performed to determine if the primary biochemical defect might be a DNA ligase. DNA-ligase activities were assayed and compared after separation of the multiple forms of ligase by AcA 34 gel-filtration chromatography of total cellular extracts. In EM9 cells the levels of the presumptive replicative forms, DNA ligase Ia (480 kd) and ligase Ib (240 kd) were about 50% and 60%, respectively, of those in the parental AA8 cells, whereas DNA ligase II (80 kd) was unaltered in EM9 . In a phenotypic revertant line ( 9R1 ) ligases Ia, Ib and II levels were 35%, 37% and 100%, respectively, of those in AA8 . The reduced levels of ligases Ia and Ib in EM9 and 9R1 cells are apparently not related directly to the mutant phenotype and may be attributable to the somewhat slower growth rates of these strains compared with those of AA8 . To determine if the repair defect in EM9 might reside in the ability to induce DNA-ligase activity after treatment with a DNA-damaging agent, AA8 and EM9 cells were treated with MMS at 30 micrograms/ml for 60 min before preparing fractions for ligase assays. Under these conditions the activities of ligases Ia and Ib decreases 70-80% in both cell lines, but ligase II increased 2.0- and 2.6-fold, respectively, in AA8 and EM9 . As a further test of defective ligase activities in EM9 , assays were performed in the presence of 0.1 M NaCl or after heating the fractions for 10 min at 50 degrees C. Although all 3 forms of ligase showed altered activity under both of these conditions, there were no significant differences between EM9 and AA8 cells. These data combined with the above results provide strong evidence that the site of the primary defect in EM9 is not in either of the DNA ligases .     

Radiation enhancement of the efficiency of DNA-mediated gene transfer in CHO UV-sensitive mutants

We have employed the Chinese hamster ovary (CHO) UV-sensitive mutant cell lines, UV5 and UV20, to determine whether ionizing and ultraviolet irradiation enhance the efficiency of DNA-mediated gene transfer in cells deficient in excision repair. Confluent AA8 (wild type), UV5, and UV20 cells were transfected (via polybrene and dimethyl sulfoxide treatments) with the recombinant DNA plasmid, pSV2-gpt, trypsinized, irradiated with either X rays or ultraviolet in suspension, and then plated into flasks. After a 48-h expression time, cells were trypsinized, counted, and plated in XMAT media to select for pSV2-gpt transformation. We report that X-ray irradiation enhances gene transfer in wild-type AA8 and in both UV-sensitive cell lines. Ultraviolet irradiation enhances gene transfer in AA8 and UV20, but not in UV5. Since both UV20 and UV5 are deficient in excision repair, we suggest that ultraviolet-enhanced gene transfer may involve a postreplication repair mechanism deficient in UV5.     

Effects of cycloheximide on protein, RNA and DNA synthesis in cultures of CHO cells and human diploid fibroblasts]

In CHO cell line and primary human diploid fibroblasts culture an incorporation of protein, RNA and DNA biosyntheses precursors was investigated under different conditions of inhibition of translation by cycloheximide (CHM). Both CHO and human fibroblasts transitory treatment by CHM in the serumfree medium resulted in inhibition of protein and DNA syntheses during S-period while RNA synthesis increased up to 130% (CHM concentration from 0.003 to 2 Mg/ml), as well as in Go--an incorporation of 3H-U increased to 200% (CHM concentration-100 Mg/ml). Long-term treatment (48 hours) in the serum-free medium resulted in decreased uptake of 3H-T and 3H-L during first 6 hours of experiment, while incorporation of 3H-U increased to 160%. By 16-th hour of treatment characters of protein, RNA and DNA syntheses came back to control levels.     

Replication of mitochondrial DNA in the early development of Misgurnus fossilis

Mitochondria isolated from Misgurnus fossilis embryos at various developmental stages were incubated with ³H-dTTP in vitro and the incorporation into mtDNA was determined. It has been found that the rate of mtDNA labeling increases exponentially with a doubling time of 7 hr from 0.01 pmole of ³H-dTMP/mg protein/hr in mitochondria from unfertilized eggs to 0.4 pmoles of ³H-dTMP/mg protein/hr in mitochondria of 35 hr embryos. The pool of intramitochondrial dTTP decreases 2.5 times during the first 10 hr after fertilization, then remains practically constant up to 35 hr of development. The rate of exogenous ³H-dTTP incorporation into the acid soluble pool of isolated mitochondria at two stages is approximately proportional to the pool size. Thus identical specific activities of ³H-dTTP inside mitochondria would be obtained even with pools of different sizes. We conclude that the increase of ³H-dTMP incorporation into mtDNA in development reflects genuine activation of mtDNA synthesis. As early as 6 hr after fertilization the bulk of the label incorporated into mtDNA is found in the fraction associated with covalently closed molecules. This pattern of labeling characteristic for replicating mtDNA is maintained throughout early development. In contrast such preferential label incorporation into the closed circular fraction was not found with mitochondria of unfertilized eggs. Closed mtDNA from unfertilized eggs contains not more than 1% of molecules with D-loops. In 35 hr embryos the corresponding value is equal to about 4%. Activation of mtDNA replication in embryogenesis is probably due to the activation of mechanisms responsible for the generation of primers for replication. DNA polymerase activity solubilized from mitochondria remains unchanged in the course of embryogenesis.     

DNA-mediated cotransfer of excision repair capacity and drug resistance into chinese hamster ovary mutant cell line UV-135.

We have investigated DNA-mediated transfer of aminopterin resistance conferred by plasmid and UV resistance conferred by genomic DNA to the Chinese hamster ovary (CHO) cell line UV-135, a UV-sensitive mutant defective in nucleotide excision repair. Plasmid pSV2gpt-CaPO4 coprecipitates induced aminopterin resistance with equal efficiency in the 6-thioguanine-resistant, aminopterin-sensitive, repair-proficient parental line AA8-4(tg-1) and in UV-135(tg-2). Genetic and molecular evidence for genomic DNA-mediated transformation of UV-135(tg-2) cells with a putative excision repair gene were obtained by demonstrating that: (i) UV resistance transformation is dependent upon and specific for genomic DNA from excision repair-competent CHO cells: (ii) UV and drug coresistant colonies are bona fide transferants as verified by hybridization and Southern blotting analysis of pSV2gpt sequences in their genomic DNAs: (iii) confirmed transferants exhibit partial to near normal UV resistances for colony formation: and (iv) UVr transferants have near normal levels of excision repair capacity. The overall frequency of drug and UV resistance cotransformation was 8 X 10(8) per cell plated. This frequency was ca. 200- to 500-fold greater than that expected from coincident but independent UVr reversion and plasmid gene transfer events. DNA transfer techniques with this CHO system will be useful for further analysis of the essential structural DNA sequences, gene cloning, and expression of functional excision repair genes.     

Characterization of CHO XPF mutant UV41: influence of XPF heterozygosity on double-strand break-induced intrachromosomal recombination

The UV hypersensitive CHO cell mutant UV41 is the archetypal XPF mammalian cell mutant, and was essential for cloning the human nucleotide excision repair (NER) gene XPF by DNA transfection and rescue. The ERCC1 and XPF genes encode proteins that form the heterodimer responsible for making incisions required in NER and the processing of certain types of recombination intermediates. In this study, we cloned and sequenced the CHO cell XPF cDNA, determining that the XPF mutation in UV41 is a +1 insertion in exon 8 generating a premature stop codon at amino acid position 499; however, the second allele of XPF is apparently unaltered in UV41, resulting in XPF heterozygosity. XPF expression was found to be several-fold lower in UV41 compared to its parental cell line, AA8. Using approaches we previously developed to study intrachromosomal recombination in CHO cells, we modified UV41 and its parental cell line AA8 to allow site-specific gene targeting at a Flp recombination target (FRT) in intron 3 of the endogenous adenine phosphoribosyltransferase (APRT) locus. Using FLP/FRT targeting, we integrated a plasmid containing an I-SceI endonuclease sequence into this site in the paired cell lines to generate a heteroallelic APRT duplication. Frequencies of intrachromosomal recombination between APRT heteroalleles and the structures of resulting recombinants were analyzed after I-SceI induction of site-specific double-strand breaks (DSBs) in a non-homologous insertion contained within APRT homology. Our results show that I-SceI induced a small proportion of aberrant recombinants reflecting DSB-induced deletions/rearrangements in parental, repair-proficient AA8 cells. However, in XPF mutant UV41, XPF heterozygosity is responsible for a similar, but much more pronounced genomic instability phenotype, manifested independently of DSB induction. In addition, gene conversions were suppressed in UV41 cells compared to wild-type cells. These observations suggest that UV41 exhibits a genomic instability phenotype of aberrant recombinational repair, confirming a critical role for XPF in mammalian cell recombination.     

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.     

Inhibiting the apoptosis pathway using MDM2 in mammalian cell cultures

The ability to regulate apoptosis in mammalian cell cultures represents one approach to developing more economical and efficient processes. Genetic modification of cells using anti-apoptotic genes is one method that may be used to improve cellular performance. This study investigates a method to inhibit upstream apoptosis pathways through the overexpression of MDM2, an E3 ubiquitin ligase for p53. Both 293 and CHO cells expressing MDM2 were examined under both batch and spent media conditions. For batch cultures, MDM2 overexpression increased viable cell densities and viabilities over control cells with the largest enhancements observed in CHO cells. When CHO cells were passaged without medium exchange, cells expressing MDM2 reached a viable cell density that was nearly double the control and survived for an extra day in culture. When exposed to spent media initially, both 293-MDM2 and CHO-MDM2 cells continued to grow for 2 days while the control cells stopped growing after the first day. DNA analysis using flow cytometry confirmed that while CHO controls were found to be undergoing DNA fragmentation, CHO-MDM2 cells exhibit DNA degradation at a much slower rate. When compared to Bcl-2-expressing cells, MDM2 expression showed greater protection against apoptosis in passaged culture, spent medium, and following transient p53 overexpression. However, expression of the RING sequence of MDM2 responsible for E3 ligase activity without the other components of the protein was found to be toxic to 293 cells in culture. These results suggest that the overexpression of heterologous MDM2 represents a promising method to delay apoptosis in mammalian cell cultures.     

Clastogenicity, photo-clastogenicity or pseudo-photo-clastogenicity: Genotoxic effects of zinc oxide in the dark, in pre-irradiated or simultaneously irradiated Chinese hamster ovary cells

Zinc oxide (ZnO), a widely used ingredient in dermatological preparations and sunscreens, is clastogenic in vitro, but not in vivo. Given that ZnO has an approximately four-fold greater clastogenic potency in the presence of UV light when compared with that in the dark, it has been suggested to be photo-clastogenic. In order to clarify whether this increased potency is a genuine photo-genotoxic effect, we investigated the clastogenicity of ZnO (mean particle size, 100 nm) in Chinese hamster ovary (CHO) cells in the dark (D), in pre-irradiated (PI, i.e. UV irradiation of cells followed by treatment with ZnO) and in simultaneously irradiated (SI, i.e. ZnO treatment concurrent with UV irradiation) CHO cells at UV doses of 350 and 700 mJ/cm(2). The cytotoxicity of ZnO to CHO cells under the different irradiation conditions was as follows: SI>PI>D. In the dark, ZnO produced a concentration-related increase in chromosome aberrations (CA). In PI or SI CHO cells, ZnO was clastogenic at significantly lower concentrations (approximately two- to four-fold) when compared with effective concentrations in the dark, indicating an increased susceptibility of CHO cells to ZnO-mediated clastogenic effects due to UV irradiation per se. The incidence of CA in SI or PI cells was generally higher than that in the dark. At similar ZnO concentrations, SI conditions generally produced higher CA incidence than PI conditions. However, when ZnO concentrations producing similar cytotoxicity were compared, CA incidences under PI or SI conditions were nearly identical. The modest increase in the clastogenic potency of ZnO following UV irradiation contrasts with the results observed with genuine photo-clastogenic agents, such as 8-MOP, which may produce an increase in clastogenic potency of >15,000-fold under SI conditions. Our results provide evidence that, under conditions of in vitro photo-clastogenicity tests, UV irradiation of the cellular test system per se may produce a slight increase in the genotoxic potency of compounds that are clastogenic in the dark. In conclusion, our data suggest that minor increases in clastogenic potency under conditions of photo-genotoxicity testing do not necessarily represent a photo-genotoxic effect, but may occur due to an increased sensitivity of the test system subsequent to UV irradiation.     

Excision repair of UV damage in human fibroblasts reversibly permeabilized by lysolecithin

We have examined nucleotide excision repair synthesis in confluent human diploid fibroblasts permeabilized with lysolecithin. Following a UV dose of 12 J/m2, maximal incorporation of [alpha 35S]dNTPs occurred at a lysolecithin concentration (approximately 80 micrograms/ml) where slightly more than 90% of the cells were initially permeable to trypan blue. However, autoradiography of cells, permeabilized at this lysolecithin concentration, demonstrated that only about 20% of the total cell population incorporated significant levels of 35S into DNA. This result presumably reflected the fact that approximately 20% of the total cell population remained permeable for much longer periods of time (up to 2 h) than the remaining cell population (less than 20 min). The incorporation of dNTPs by UV-irradiated, permeabilized cells appeared to be bona fide excision repair synthesis since: (1) Incorporation was completely absent in unirradiated, permeabilized cells and in irradiated, permeabilized repair-deficient cells. (2) Nucleotides incorporated in the presence of BrdUTP were associated with normal density DNA. (3) The apparent Km for all 4 dNTPs was 50-100 nM, in agreement with past reports on human fibroblasts irreversibly permeabilized by cell lysis. (4) DNA associated with the newly incorporated dNTPs underwent ligation and rearrangements in chromatin structure analogous to what is observed in intact human cells. Repair incorporation of dNTPs was rapid and linear during the first 2 h after UV irradiation and permeabilization. After this time, incorporation ceased or continued at a much slower rate. Cell viability experiments and autoradiography demonstrated that the cells permeabilized to [3H]dNTPs were capable of carrying out DNA replication and cell division. Thus, confluent human diploid fibroblasts can be reversibly permeabilized to labeled dNTPs by lysolecithin for the study of excision repair following physiologic doses of UV radiation. However, under these conditions, only a fraction of the cells remain permeable for an extended period of time.