DNA Of Chinese Hamster Cells Research Articles

Chromosome damage induced by restriction endonucleases recognizing thymine-rich DNA sequences in electroporated CHO cells.

The influence of BrdU substitution of DNA in Chinese hamster cells on the frequencies of chromosomal aberrations induced by three restriction endonucleases which recognize thymine-rich sequences in DNA has been studied. The restriction enzymes chosen were Eco RI (recognition site G/AATTC), Sca I (AGT/ACT), and Dra I (TTT/AAA). A restriction enzyme that does not have thymine in the recognition sequence, Hae III (GG/CC), was also tried. These enzymes were introduced into cells by electroporation after two cell cycles of BrdU substitution and the aberration yields compared with that observed in non-substituted cells. Our results seem to indicate that the BrdU-substituted chromatin becomes resistant to the chromosome-breaking activity of the restriction enzymes recognizing thymine-rich DNA sequences. These observations are compared with the patterns of cutting of isolated DNA as shown by agarose gel electrophoresis.

Measurement of Double-Strand Breaks in Chinese Hamster Cell DNA by Neutral Filter Elution: Calibration by 125 I Decay

We labeled the DNA of Chinese hamster lung V79 cells with 125I in the form of iododeoxyuridine and subsequently measured the elution of the DNA through polycarbonate filters at pH 9.6 and pH 7.2. Since decay of incorporated 125I produces predominantly double-strand breaks (DSB) in DNA at a rate close to one DSB per 125I decay, this measurement provides an absolute calibration for the assay of DSBs by neutral filter elution. Neutral elution profiles are not first order with respect to elution time; thus we have examined the relationships between accumulated 125I decays and several functions of retention of DNA on the filter at various times during the elution process. At both pH 9.6 and pH 7.2 there were linear relationships between accumulated decays and certain retention functions. The retention function most closely correlated to 125I decays for both pH values was the logarithm of the ratio of the retention of control DNA to that of 125I-labeled DNA, both evaluated at the 9th fraction (13.5 h of elution). The linear relationship between this ratio and 125I decays allows DSB induction to be determined directly from retention values. The calibration was used to measure DSBs induced by X rays.

Genetic toxicology of the photosensitization of Chinese hamster cells by phthalocyanines.

Chloroaluminum phthalocyanine (CAPC) was recently shown to photosensitize cell killing in culture and tumor destruction in vivo. Because this compound is potentially useful in the photodynamic therapy of cancer, its properties as a genotoxic agent were evaluated. Applying the technique of alkaline elution to study DNA integrity, it was found that CAPC could produce single‐strand breaks in the DNA of Chinese hamster cells after exposure to white fluorescent light. At equicytotoxic doses, the number of DNA strand breaks produced by CAPC photosensitization was about three times lower than that induced by X‐irradiation. During incubation in growth medium after exposure to CAPC‐plus‐fluorescent light, cells rejoined DNA strand breaks at a rate similar to that observed after X‐irradiation. Resistance to 6‐thioguanine (6‐TG') or to ouabain (OUA') were used as end points of mutagenic potential. Following a treatment that caused ‐90% cell killing, there was a slight mutagenic effect, i.e. the frequencies were increased by ‐40% above the background or spontaneous mutations. However, this enhancement was not statistically significant. Taken together, the foregoing, plus an earlier observation that there is no variation in the sensitivity of cells to CAPC + light through the cell cycle, lead to the inferences that DNA damage does not play a major role in cell killing and that the mutagenic potential of this treatment is small.

Chromosome damage induced by decay of 3H and 125I incorporated into DNA of Chinese hamster cells

The present study was undertaken to compare the frequency of chromatid-type aberrations in Chinese hamster cells with previous results on accumulation of unrepaired DNA-strand breaks after incorporation of 3H-TdR or 125IUdR into DNA. A linear-quadratic function was fitted by the weighted-least-square method to the data on yield of chromatid aberrations at different dpm values. Based on a significant linear response at low doses, RBE for 125I in relation to 3H was calculated for (i) chromatid breaks (17 ± 6), (ii) the sum of isochromatid breaks and chromatid exchanges (21 ± 9), and (iii) the total number of chromatid aberrations (18 ± 5). Analogously, the RBE for accumulation of DNA-strand breaks was determined (13 ± 6). Our results are consistent with the assumption that chromosomal aberrations mainly originate from unrepaired DNA-strand breaks.

Role of mitochondrial DNA in cell death induced by 125I decay.

The role of mitochondrial DNA in radiation-induced cell death was determined by selective [125I]iododeoxyuridine (125IUdR) incorporation into exclusively nuclear sites compared to labelling in both nuclear and mitochondrial DNA of Chinese hamster cells. Such selectivity was achieved by using berenil (25 micrograms/ml for 24 h), a drug which inhibits mitochondrial DNA synthesis without affecting incorporation of 125IUdR into nuclear DNA but does not result in reduced clonogenicity or cell cycle perturbations or alteration in the X-ray response of cells. There was no difference in cell killing between cells with nuclear labelling alone compared with nuclear plus mitochondrial labelling. The absence of decays in mitochondrial DNA does not affect the ability of 125I to induce lethal cell damage. The two treatment groups have superimposable curves with a D0 of 96 decays/cell. These findings indicate that mitochondrial DNA is not the most sensitive target for radiation-induced cell death from 125I decay.

The induction of SCE with relation to specific base methylation of DNA in Chinese hamster cells by N-methyl-N-nitrosourea and dimethylsulfate.

The relationship between the formation of alkylated purines in DNA and sister chromatid exchange (SCE) induction has been studied. Both exponentially growing and density-inhibited Chinese hamster (V79) cultures were treated with various doses of N-methyl-N-nitrosourea (MNU) or dimethylsulfate (DMS). The colony-forming ability and induced frequencies of SCEs were assayed. Following the exposure of density-inhibited cells to radiolabeled methylating agents these phenomena were related to the levels of 7-methylguanine (7-meGua), 0(6)-methylguanine (0(6)-meGua) and 3-methyladenine (3-meAde) in the DNA. At equitoxic doses MNU and DMS induced similar frequencies of SCEs. Since, at equitoxic doses MNU produces about 20 times more 0(6)-meGua in V79-cell DNA than does DMS, this indicates that the formation of this adduct in DNA is not critical for the induction of SCEs by these alkylating agents. Dimethylsulfate-induced SCEs may be mediated via the production of both 3-meAde and 7-meGua in the DNA; these 2 methylated purines may also be responsible for MNU-induced SCE. No one specific methylated purine was identified, therefore, as being solely accountable for the formation of SCEs. The repair of lesions in the DNA of nonreplicating V79 cells lead to a reduction in the SCE frequency on their subsequent release from the density-inhibited state. This suggests that excision repair is not responsible for the formation of SCEs.

DNA Damage and Radiosensitizers: M. luteus Sensitive Sites for Misonidazole-TAN Combination

It has been shown previously that TAN does not enhance production of single-strand breaks (SSB) in DNA of Chinese hamster cells irradiated under hypoxia. In contrast, misonidazole does enhance the yield of SSB, but this SSB enhancement by misonidazole is reduced if TAN is present with misonidazole during irradiation. It was also shown that each of these sensitizers enhances base/sugar damage, measured with the aid of bacterial enzymes (MLS). The results presented here for MLS damage in mammalian cells irradiated in the presence of the misonidazole-TAN combination indicate that the two drugs act independently in enhancing MLS damage, and hence they interact with different types of lesions to produce base/sugar damage. It is proposed that the protection by TAN in mammalian cells observed at the survival level is due to "repair" of some of that type of damage which would otherwise become a misonidazole-enhanced SSB.

The induction of SCE and chromosomal aberrations with relation to specific base methylation of DNA in Chinese hamster cells by N-methyl-N-nitrosourea and dimethyl sulphate.

Chinese hamster cells (V79) were treated, either as exponentially proliferating cultures or under conditions where they were density-inhibited, with various doses of the potent carcinogen N-methyl-N-nitrosourea (MNU) or the relatively weak carcinogen dimethylsulphate (DMS). The colony forming ability of these cells and the induced frequencies of sister chromatid exchanges (SCEs) and chromosomal aberrations were assayed. Following the exposure of density-inhibited cells to radio-labelled methylating agents (labelled in the methyl group) these phenomena were related to the levels of 7-methylguanine (7-meGua), O6-methylguanine (O6-meGua) and 3-methyladenine (3-me-Ade) in the DNA. At equitoxic doses MNU and DMS induced similar frequencies of SCEs and chromosomal aberrations. Since, at equitoxic doses, MNU produces approximately 20 times more O6-meGua in V79 cell DNA than does DMS, this indicates that the formation of O6-meGua in DNA is not a major cause of SCEs and chromosomal aberrations. DMS-induced SCEs may be mediated via the production of both 3-meAde and 7-meGua in the DNA; these two methylated purines may also be responsible for MNU-induced SCEs. Therefore, no one specific methylated purine was identified as being solely accountable for the formation of SCEs. Also, the repair of lesions in the DNA of non-replicating V79 cells leads to a reduction in the SCE frequency on their subsequent release from the density-inhibited state, suggesting that repair is not intimately responsible for their formation. No association was discernable between chromosomal aberrations and any of the three methylated purines studied.

Effect of Oxygen-Radiosensitizer Mixtures on the Radiation Response of Chinese Hamster Cells, Line V-79-753B, in Vitro: II. Determination of the Initial Yield of Single-Strand Breaks in the Cellular DNA Using a Rapid Lysis Technique

The present data show that the maximum yield of single-strand breaks (ssb) in the cellular DNA of Chinese hamster cells V-79-753B is produced at a concentration of oxygen that produces an enhancement ratio for cell survival of 1.9. The relationship between the oxygen concentration and enhancement ratio for survival in this cell line is biphasic with a plateau at ER = 1.9 over the range of 1.5 to 7 ~M 02. For concentrations of oxygen below 1.5 pM a linear relationship between 1/Do and the initial yield of ssb is found. Electron affinic and free radical radiosensitizers operate by different mechanisms which are reflected at the level of ssb production; electron affinic compounds increase the yield of ssb in anoxia and in the presence of low concentrations of oxygen, whereas free radical radiosensitizers do not. The observation that TMPN can compete with oxygen or misonidazole in reactions that lead to changes in radiosensitivity but not ssb production indicates that the relationship between the two parameters must be casual and not causal.

The Excision of N‐Methyl‐N‐nitrosourea‐Induced Lesions from the DNA of Chinese Hamster Cells as Measured by the Loss of Sites Sensitive to an Enzyme Extract that Excises 3‐Methylpurines but not O6‐Methylguanine

An enzyme extract from Micrococcus luteus excises 3-methyladenine and 3-methylguanine but not O6-methylguanine, 7-methylguanine, 1-methyladenine or 7-methyladenine from DNA reacted with N-methyl-N-nitrosourea. The extract was used to detect lesions in the DNA of Chinese hamster cells treated in culture with N-methyl-N-nitrosourea. It was concluded that 3-methyladenine is excised from these cells with a half-life of about 2.3 h.

23 Excision repair of cis platinum II diammine dichloride induced damage to DNA of chinese hamster cells

23 Excision repair of cis platinum II diammine dichloride induced damage to DNA of chinese hamster cells

Direct evidence for the role of incorporated BUdR in the induction of sister chromatid exchanges

Abstract The level of bromodeoxyuridine (BUdR) substitution for thymidine (dThd) in nascent DNA of Chinese hamster cells increases rapidly with increasing BUdR concentration at low BUdR/base pair (BU/bp) ratios and then increases much more slowly at higher ratios. The relationship between percentage substitution and BU/bp indicates that BUdR competes with dThd for incorporation into DNA and that BUdR concentration has no effect on the effective dThd/base pair ratio in these cells. Equal amounts of BUdR are incorporated during both the first and second cell cycles (S phases) after provision of the dThd analog. Incorporation of BUdR induces sister chromatid exchanges (SCEs), and induction appears to occur during the cell cycle (S phase) in which incorporation occurs, with incorporated BUdR from previous cycles having little effect. BUdR-induced SCE frequencies are linearly proportional to the percentage substitution of BUdR for dThd. Extrapolation to zero substitution indicates that SCEs occur spontaneously at a frequency of 0.045 per chromosome per cycle, and that many so-called “spontaneous” SCEs are actually induced by BUdR.

Repair of alkylated DNA in Chinese hamster cells measured by loss of enzyme-sensitive sites in isolated DNA

Lesions introduced into the DNA of Chinese hamster cells by dimethyl sulphate (DMS) are recognized by an enzyme(s) present in crude extracts of M. luteus . Such lesions are rapidly lost from DNA during post treatment incubation of cells. The number of these enzyme-sensitive sites in methylated DNA, their rate of loss and their heat lability are consistent with the majority being 3-methyladenine adducts in DNA.

Post-replication repair of 7-bromomethyl benz(a)anthracene damaged DNA in Chinese hamster cells: J.J. Roberts, H.W. van den Burg, F. Friedlos and D. Kirkland, Institute of Cancer Research: Royal Cancer Hospital, Pollards Wood Research Station, Nightingales Lane, Chalfont St. Giles, Bucks HP8 4SP (U.K.)

Post-replication repair of 7-bromomethyl benz(a)anthracene damaged DNA in Chinese hamster cells: J.J. Roberts, H.W. van den Burg, F. Friedlos and D. Kirkland, Institute of Cancer Research: Royal Cancer Hospital, Pollards Wood Research Station, Nightingales Lane, Chalfont St. Giles, Bucks HP8 4SP (U.K.)

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 radio-activity 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.

Effect of radioprotective aminothiols on the induction and repair of single-strand breaks in the DNA of irradiated mammalian cells.

The effect of cysteamine and 2-mercapto-propionyl-glycine on the induction and repair of single-strand breaks in the DNA of Chinese hamster cells by irradiation in vitro in the presence of oxygen or in anoxia was investigated. The substances protected against the formation of breaks by irradiation both under oxic and anoxic conditions. The substances also exhibited a toxic effect which resulted in degradation of DNA. Cysteamine inhibited the rejoining of breaks after radiation exposure.

Fragmentation of chromatin with 125I radioactive disintegrations

The DNA in Chinese hamster cells was labeled first for 3 h with [3H]TdR and then for 3 h with [125I]UdR. Chromatin was extracted, frozen, and stored at -30 degrees C until 1.0 X 10(17) and 1.25 X 10(17) disintegrations/g of labeled DNA occurred for 125I and 3H respectively. Velocity sedimentation of chromatin (DNA with associated chromosomal proteins) in neutral sucrose gradients indicated that the localized energy from the 125I disintegrations, which gave about 1 double-strand break/disintegration plus an additional 1.3 single strand breaks, selectively fragmented the [125I] chromatin into pieces smaller than the [3H] chromatin. In other words, 125I disintegrations caused much more localized damage in the chromatin labeled with 125I than in the chromatin labeled with 3H, and fragments induced in DNA by 125I disintegrations were not held together by the associated chromosomal proteins. Use of this 125I technique for studying chromosomal proteins associated with different regions in the cellular DNA is discussed. For these studies, the number of disintegrations required for fragmenting DNA molecules of different sizes is illustrated.

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.

Detection by density equilibrium centrifugation of recombinant-like DNA molecules in somatic mammalian cells.

When the nuclear DNA of Chinese hamster cells grown in the presence of bromo-deoxyuridine for one or less generation is analysed in an alkaline CsCl density gradient, a small fraction of replicated DNA can be found at densities intermediate between those of the light and heavy strands. The labelling pattern of these displaced molecules and their segregation into fully light and heavy fragments after sonication indicate that they are formed by the covalent joining of light and heavy segments. The duplexes giving rise to displaced DNA upon denaturation are mostly found at the position of light-heavy DNA in a neutral gradient; a minor component, however, bands at densities between light-heavy and heavy-heavy DNA. The high degree of renaturability of the former molecules suggests that they contain cross-links between the complementary strands; the latter, on the contrary, are likely to comprise a segment substituted in both strands, which is interpreted as being a heteroduplex resulting from somatic crossing-over between uninemic sister chromatids. Although the frequency of these recombinant-like molecules is increased several-fold after u.v. irradiation, the data do not support any direct implication of these exchanges in a postreplication repair mechanism of the type found in bacteria.

Hybridization of simian virus 40 complementary RNA with nucleolus-associated DNA isolated from simian virus 40-transformed Chinese hamster cells

Nucleolus-associated DNA was isolated from SV40-transformed Chinese hamster cell lines, and hybridized with SV40 [ 3H]cRNA to determine whether a unique integration site could be selectively fractionated between nucleoli and total nuclear DNA. The distribution pattern of integrated SV40 genomes between these two DNA fractions varied from clone to clone. These results are consistent with multiple sites of integration of SV40 DNA into Chinese hamster cell DNA.