Inhibition of post-replication repair of alkylated DNA by caffeine in Chinese hamster cells but not HeLa cells

Caffeine has been found to potentiate the lethal effects of sulphur mustard (SM) and N-methyl-N-nitrosourea (MNU) in a line of Chinese hamster cells but not in a line of HeLa cells. The sensitization of SM-treated cells by caffeine was S phase specific, and persisted for up to 24 h after alkylation of asynchronous cell cultures. The sensitization of MNU-treated cells, however, was not S phase specific but persisted for up to 50 h after the initial alkylation. Possible explanations for this difference between these two types of alkylating agent were discussed. Previously, evidence was presented which suggested that the alkylation-induced delay in the time of the peak rate of DNA synthesis in Chinese hamster cells was associated with the operation of post-DNA replication repair mechanism in these cells. Caffeine has now been found to reverse this alkylation-induced delay of DNA synthesis in both SM- and MNU-alkylated Chinese hamster cells. It is therefore proposed that caffeine sensitizes alkylated cells by inhibition of a post-replication DNA repair mechanism. No support was obtained for the alternative possibility that caffeine inhibits alkylation-induced excision repair of damaged DNA. The role of DNA repair in the production of the lethal mutagenic and cytological effects of alkylating agents is discussed.     

Post-replication repair of DNA in Chinese hamster cells treated with cis platinum (II) diamine dichloride. Enhancement of toxicity and chromosome damage by caffeine.

The anti-tumor agent cis platinum (II) diammine dichloride (cis Pt(II)) caused chromosomal abnormalities in Chinese hamster V79-379A cells. The time of appearance of these abnormalities suggested that they arise as a consequence of DNA synthesis on a damaged template. The yield and severity of chromosomal abnormalities was greatly enhanced by a non-toxic concentration of caffeine, and this enhancement was associated with a potentiation of cis Pt(II) induced cell death. These results suggest that damage to DNA which arises from cis Pt(II) treatment can be repaired in this cell line by a caffeine-sensitive post-replication repair process.     

Inhibition of repair of UV-damaged DNA by caffeine and mutation induction in Chinese hamster cells

The effect of caffeine on UV-irradiated Chinese hamster cells in vitro was studied on the cellular and molecular levels. Caffeine (1 mM) was shown to decrease the colony-forming ability and the frequencies of spontaneous and UV-induced mutations in Chinese hamster cells. The effect of caffeine in reducing the frequency of UV-induced mutations was demonstrated only if caffeine was present in the culture medium during the first post-irradiation cell division. Using alkaline sucrose gradient centrifugation, both parental and newly synthesized DNA in UV-irradiated and unirradiated cells were studied in the presence and absence of caffeine. Caffeine affected the sedimentation profile of DNA synthesized in UV-irradiated cells but not in unirradiated cells. Caffeine had no apparent effect on the incorporation of [³H]-thymidine into DNA of control or UV-irradiated cells, nor on the small amount of excision of UV-induced pyrimidine dimers. These results may be interpreted by a hypothesis that caffeine inhibits a certain S-phase specific, post-replication, dark-repair mechanism. The hamster and perhaps other rodent cells exposed to low doses of UV are capable of DNA replication, by-passing the non-excised pyrimidine dimers. This postulated repair process probably involves de novo DNA synthesis to seal the gaps in the nascent strand. This repair may be also responsible for the enzymatic production of mutations.     

Interaction of caffeine with the DNA of Chinese hamster cells.

Incubation of Chinese hamster cells with labelled caffeine leads to transfer of radioactivity to DNA. This association occurs during the S phase of the cell cycle and involves parental as well as newly synthesised strands. The replacement of thymidine by BrdUrd prevents the incorporation radioactivity from caffeine into the DNA strands containing BrdUrd. Thymine is the only base which becomes labelled and data suggesting the participation of methyl groups of caffeine in the biosynthesis of thymine are presented. Ultraviolet irradiation increases the incorporation of radioactivity from caffeine to DNA.     

Inhibition of repair of radiation-induced DNA damage by thermal shock in Chinese hamster ovary cells

The effect of exposure to elevated temperatures (41-45 degrees C) on the repair of radiation-induced DNA strand breaks was measured in monolayer cultured Chinese hamster ovary (CHO) cells. Prior exposure of cells to temperatures between 43 and 45 degrees C resulted in significant decreases in the rate of repair of DNA damage. Exposure to 45 degrees C for 15 min slowed the rate of DNA repair to 0.17 of the control repair rate. The To for inactivation of DNA repair was observed to be 34, 13 and 6 min at 43, 44 and 45 degrees C, respectively. Stepdown-heating (45 degrees C for 15 min followed by repair at 41 degrees C) resulted in greater inhibition of DNA repair (0.11 of the control rate) than was observed after acute heating alone. Repair at 41 degrees C was observed to proceed in unheated cells at a faster rate than at 37 degrees C. An Arrhenius analysis of the inactivation kinetics of DNA repair between 43 and 45 degrees C indicated an activation energy of 140 kcal mol-1 of protein for the inhibition of DNA repair. In general, the results were inconsistent with either a retardation of the DNA repair rate or an increase in unrepaired DNA lesions being responsible for heat-induced radiosensitization.     

Bufalin influences the repair of X-ray-induced DNA breaks in Chinese hamster cells

The bufadienolide bufalin, a component of the Chinese medicine chan'su, has been reported to selectively inhibit the growth of various lines of human cancer cells, due at least in part to its specific effect on topoisomerase (topo) II. We have treated Chinese hamster ovary (CHO) cells with doses of bufalin that result in a dramatic reduction in both the level and catalytic activity of topo II without any concomitant induction of DNA damage, as assessed by the comet assay. When cells were pre-treated with bufalin and then irradiated with X-rays, a follow-up study revealed that the kinetics of DNA repair was clearly affected, with a general delay in the restoration of DNA to the situation observed in non-irradiated controls. The possible involvement of topo II in radiation damage repair is discussed.     

Preferential binding of cisplatin to mitochondrial DNA of Chinese hamster ovary cells

Some chemical carcinogens localize preferentially in mitochondrial DNA (mtDNA) when compared with genomic DNA (gDNA). Here we compare the ability of cisplatin (cis-diamminedichloroplatimum[II]) to induce DNA adducts in both genomic and mtDNA of Chinese hamster ovary (CHO) cells in culture. Cytotoxicity was examined by cell survival 4, 8 and 24 h afer exposure to 50 μM cisplatin. Cisplatin-DNA adducts were measured in DNA from nuclear and mitochondrial fractions by dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA), a sensitive competitive microtiter-based immunoassay utilizing antiserum elicited against cisplatin-modified DNA. An additional comparison of cisplatin-DNA binding in both compartments was performed by immunoelectron microscopy using the cisplatin-DNA antiserum and colloidal gold. DELFIA analysis of cisplatin-DNA adducts in gDNA and mtDNA showed a six-fold higher incorporation of drug into mtDNA as compared to gDNA. Morphometric studies of colloidal gold distribution in photomicrographs of CHO cells showed mtDNA to contain a four-fold higher concentration of cisplatin as compared to nuclear DNA. Therefore, both methods demonstrated a preferential binding of cisplatin to mtDNA versus gDNA.     

Inducible repair of DNA single-strand breaks in gamma-irradiated Chinese hamster cells

Preirradiation of Chinese hamster cells with low-level UV-light does not influence the efficiency of repair of gamma-radiation-induced DNA single-strand breaks. With fractionated gamma-irradiation, cycloheximide delivered during the interval between the two fractions reduces the number of DNA breaks (compared to that in cells affected by the same nonfractionated dose). The data obtained indicate the presence of an inducible component of repair of DNA single-strand breaks in gamma-irradiated Chinese hamster cells.     

DNA damage in Chinese hamster cells repeatedly exposed to low doses of X-rays

In a recent paper we reported the results of an experiment carried out by analysing chromosomal damage in Chinese hamster (CHO) cells exposed to low doses of X-rays. The present investigation was undertaken in order to validate those results using a different approach, the single cell gel electrophoresis assay (comet assay) immediately after irradiation. Cells were cultured during 14 cycles, irradiation treatment was performed once per cycle when the cells were at 90-95% of confluence. Doses of 2.5, 5.0 and 10.0 mSv were used. Sequential irradiation of CHO cells induced a decrease of cells without migration and an increase of cells showing DNA damage with the three doses employed. Significant increases of low-level damaged cells (p < 0.001) were found for the 14 exposures when compared to controls except for the first irradiations with 2.5 and 10 mSv, respectively. No significant increase of the frequency of cells with severe damage was observed in any case. These findings could be explained by assuming a complex interactive process of cell recovery, DNA damage and repair together with the induction of genomic instability, the incidence of bystander effects as well as some kind of radioadaptative response of the cells. If these phenomena are limited to the cell line employed deserves further investigation.     

Inhibition of human excision DNA repair by inorganic arsenic and the co-mutagenic effect in V79 Chinese hamster cells

We investigated the lethal, UV killing-potentiating and repair-inhibiting effects of trivalent arsenic trioxide (As2O3) and pentavalent sodium arsenate (Na2HAsO4) in normal human and xeroderma pigmentosum (XP) fibroblasts. The presence of As2O3 for 24 h after UV irradiation inhibited the thymine dimer excision from the DNA of normal and XP variant cells and thus the subsequent unscheduled DNA synthesis (UDS): excision inhibitions were partial, 30-40%, at a physiological dose of 1 microgram/ml and 100% at a supralethal dose of 5 micrograms/ml. Correspondingly, As2O3 also potentiated the lethal effect of UV on excision-proficient normal and XP variant cells in a concentration-dependent manner, but not on excision-defective XP group A cells. Na2HAsO4 (As5+) was approximately an order of magnitude less effective in preventing all the above repair events than As2O3 (As3+) which is highly affinic to SH-containing proteins. The above results provide the first evidence that arsenic inhibits the excision of pyrimidine dimers. Partially repair-suppressing small doses of As2O3 (0.5 microgram/ml) and Na2HAsO4 (5 micrograms/ml) enhanced co-mutagenically the UV induction of 6-thioguanine-resistant mutations of V79 Chinese hamster cells. Thus, such a repair inhibition may be one of the basic mechanisms for the co-mutagenicity and presumably co-carcinogenicity of arsenic. XP group A and variant strains showed a unique higher sensitivity to As2O3 and Na2HAsO4 killing by a yet unidentified mechanism.     

Defect in the caffeine-dependent postreplication DNA repair in UV-sensitive Chinese hamster clone cells

The postreplication repair of DNA in the presence of caffeine was investigated in the Chinese hamster clones cells of different UV-sensitivity. Caffeine (10(-2)M) inhibits the repair of daughter DNA (PRR of DNA) in the UV-light irradiated cells of UV-resistant clones CHO-K1, 14-2C-1 and V79, but does not influence the PRR of DNA in cells of UV-sensitive clones CHS1 and CHS2. Thus, deficiency of PRR of DNA in cells of UV-sensitive clones (the repair of daughter DNA is significantly retarded) is associated with the defect of the caffeine-dependent component of this repair process.     

Azacytidine-induced reactivation of a DNA repair gene in Chinese hamster ovary cells.

Six X-ray-sensitive (xrs) strains of the CHO-K1 cell line were shown to revert at a very high frequency after treatment with 5-azacytidine. This suggested that there was a methylated xrs+ gene in these strains which was structurally intact, but not expressed. The xrs strains did not complement one another, and the locus was autosomally located. In view of the frequency of their isolation and their somewhat different phenotypes, we propose that the xrs strains are mutants derived from an active wild-type gene. However, there is in addition a methylated silent gene present in the genome. Azacytidine treatment reactivated this gene. We present a model for the functional hemizygosity of mammalian cell lines, which is based on the inactivation of genes by de novo hypermethylation. In contrast to results with xrs strains, other repair-defective lines were found not to be reverted by azacytidine.     

Accumulation of low molecular weight DNA and changes in chromatin structure in HeLa cells treated with human fibroblast interferon

The addition of human fibroblast interferon (IFN-beta) (100 units/ml) at the S/G2 boundary of the cell cycle of synchronously grown HeLa cells is characterized by the accumulation of newly synthesized low molecular weight DNA and changes in chromatin assembly. In addition, there is a 3-fold stimulation in the incorporation of tracer amounts of [3H]thymidine, but not [3H] deoxyguanosine, into DNA and a 2-fold increase in the incorporation of [3H]dTTP into the DNA of isolated nuclei. Fluorescence-activated cell sorting by laser flow cytometry revealed that IFN-beta-treated cells were delayed in entering and passing through the S phase. The inhibition of proliferation of HeLa cells treated with IFN-beta is characterized by a 3-fold accumulation of newly synthesized DNA of Mr less than 56 X 10(6) compared to untreated cells as determined by alkaline sucrose gradient centrifugation. The newly synthesized DNA in IFN-beta-treated cells was replicative and not repair DNA. The observation that IFN-beta inhibits the processing of newly synthesized low molecular weight DNA into normal DNA might be explained by the intracellular accumulation of S-adenosylhomocysteine in IFN-beta-treated HeLa cells (de Ferra, F., and Baglioni, C. (1983) J. Biol. Chem. 258, 2118-2121) which could change the soluble ribonucleotide and deoxyribonucleotide pool and ultimately affect DNA processing. Interferon may also affect processing of DNA by interfering with normal chromatin assembly. Evidence for the effect of IFN-beta on chromatin assembly is provided; we have observed a more condensed structure in IFN-beta treated cells by circular dichroism spectroscopy. Simultaneous with the affect on chromatin assembly, there is a 70% decrease in poly(ADP-ribosylation) of either histone and/or non-histone proteins. The loss of coordination between the pool size for DNA synthesis, decreased postsynthetic modifications of chromatin, and normal chromatin formation may explain the inability of the cell to differentiate and to continue cell division.     

The relevance of caffeine post-treatment to SCE incidence induced in Chinese hamster cells

The influence of caffeine post-treatment on sister-chromatid exchanges (SCE) and chromosomal aberration frequencies on Chinese hamster cells exposed to a variety of chemical and physical agents followed by bromodeoxyuridine (BrdUrd) was determined. After 2 h treatment, N-methyl-N′-nitrosoguanidine (MNNG) and cis-platinum(II)diamine dichloride (cis-Pt(II)) induced a 7- and 6-fold increase in SCE, respectively, while 4-nitroquinoline-1-oxide (4NQO), methyl methanesulfonate (MMS), proflavine, and N-hydroxyfluorenylacetamide (OH-AAF) caused a 2–3-fold increase in SCE compared to controls treated with BrdUrd alone. Ultraviolet light doubled the number of SCE. The lowest increase of SCE was obtained with bleomycin and X-irradiation. Caffeine post-treatment caused a statistically significant increase in the frequency of SCE induced by UV- and X-irradiation as well as by 4NQO and MMS but did not alter the number of SCE induced by MNNG, cis-Pt(II), proflavine, OH-AAF, and bleomycin.

Caffeine post-treatment increased the number of cells with chromosomal aberrations induced by MNNG, cis-Pt(II), UV, 4NQO, MMS, and proflavine. With the exception of proflavine, these agents are dependent on DNA and chromosome replication for the expression of the chromosomal aberrations. Caffeine enhancement of cis-Pt(II) chromosomal aberrations occurred independently of the time interval between treatment and chromosome preparations. Chromosomal damage produced by bleomycin and X-irradiation, agents known to induce chromosomal aberrations independent of “S” phase of the cell cycle, as well as the damage induced with OH-AAF was not influenced by caffeine post-treatment.

The enhancement by caffeine, an inhibitor of the gap-filling process in post-replication repair, of chromosomal aberrations induced by “S” dependent agents, is consistent with the involvement of this type of repair in chromosomal aberration formation. The lack of inhibition of SCE frequency by caffeine indicates that post-replication repair is probably not important in SCE formation.