Synchronous Chinese Hamster Ovary Cells Research Articles

Condensation of Interphase Chromosomes in Nuclei of Synchronized CHO Cells

The escape of individual interphase chromosomes from nuclei of reversibly permeabilized Chinese hamster ovary (CHO) cells was utilized for the visualization of condensing interphase chromosomes in a cell cycle-dependent manner in synchronized cells. Major interphase chromosomal forms include: (a) mid-S-phase globular chromosomes at 3.0 C-value, (b) late mid-S-phase fibrous hemicircular forms (3.3 C), (c) late-S-phase supercoiled ribbons (3.7 C), and (d) end-S-phase elongated, bent prechromosomal structures (4.0 C).

Cell cycle-dependent expression of the CHO2 antigen, a minus-end directed kinesin-like motor in mammalian cells

A 69 kDa protein present in the interphase centrosome and mitotic spindle/pole was identified with the CHO2 monoclonal antibodies raised against mitotic spindles isolated from Chinese hamster ovary (CHO) cells. Isolation and characterization of antigen-specific cDNAs and recombinant proteins demonstrated that the protein is a minus-end-directed microtubule motor with the motor domain located at the C terminus. Affinity-purified polyclonal antibodies prepared to bacterially expressed fusion proteins revealed the presence of the antigen in interphase nuclei, and the degree of nuclear immunostaining intensity varied among cells at different cell cycle stages. In order to examine the change of antigen expression during the cell cycle, we prepared synchronized populations of CHO cells, double stained with CHO2 and PCNA (proliferating cell nuclear antigen) antibodies, and quantitated the amounts of nuclear fluorescence using the MetaMorpho image analysis software package. Cells right after cell division contained nuclei with the lowest level of CHO2 immunofluorescence. The immunofluorescence intensity progressively increases through G1 to S, reaching a maximum level by the end of G2. The antibody uniformly stained the entire nuclear region, and the total amount of fluorescence detected in G2 cells was greater than three times that of G1 cells. Cell cycle dependent accumulation of the CHO2 antigen was further confirmed by immunoblot analysis of the protein included in whole cell extracts and nuclei isolated from synchronized CHO cells. Northern blot analysis showed that, although the CHO2-transcript accumulated during later stages of the cell cycle, its abundance declined through G1 to S, and was lowest in cells at the early S phase. The difference in the expression pattern of the antigen protein and its transcript may suggest the presence of multiple mechanisms controlling the level of CHO2 antigen during the course of the cell cycle.

Characterization of the DNA damage in 6-thioguanine-treated cells

6-Thioguanine (TG) incorporation into DNA has been associated with cytotoxicity and DNA damage in Chinese hamster ovary (CHO) and murine leukemia L1210 cells. According to alkaline elution analysis, single-strand breaks (SSB) occur in both cell types. DNA-protein and interstrand crosslinks are prominent features of TG effects in L1210, CEM, and HL-60 but not CHO cells. To assess which DNA strand experiences SSB in CHO cells, the cells were synchronized by growth to confluence (late g 1, S). The cells were then diluted into fresh medium so that they underwent a round of division during a subsequent 16-hr interval. They were treated with TG during this first cell cycle, and mitotic cells were harvested at the end of the first cycle using colcemid. SSB were determined in parental DNA (radiolabeled with thymidine during growth to confluence), TG-containing DNA (radiolabeled with [ 14C]TG during drug exposure), and daughter DNA (labeled with thymidine during the second cell cycle). SSB occurred in TG-containing DNA late in the second cell cycle after drug exposure and in the DNA synthesized from a TG-DNA template (daughter DNA). This observation is consistent with the known delayed cytotoxicity and chromosomal aberrations seen in CHO cells. The SSB suggest relatively normal elongation of DNA containing TG but altered synthesis and/or ligation from a TG-DNA template. This premise was tested in synchronized CHO cells. The DNA strand incorporating TG elongated naturally; however, DNA elongation was impaired in the cell cycle following TG treatment. The results are consistent with SSB in daughter DNA synthesized from a TG-DNA template due to inability to elongate the newly-synthesized strand.

Cell Killing, Chromosomal Aberrations, and Division Delay as Thermal Sensitivity Is Modified during the Cell Cycle

Synchronous Chinese hamster ovary cells were treated in G1 or S phase with cycloheximide or procaine hydrochloride before and during heating at 43 degrees C. Cycloheximide and procaine apparently act by different mechanisms, with cycloheximide inhibiting protein synthesis and procaine hydrochloride supposedly affecting cellular membranes. Both agents, however, modify the heat damage expressed as chromosomal aberrations, cell killing, or division delay. Furthermore, the approximately twofold protection with cycloheximide treatment or twofold sensitization with procaine treatment is the same for the three end points and for heating during either G1 or S phase. However, heat induces chromosomal aberrations observed in metaphase when cells are heated in S phase but not when they are heated in G1. Finally, for the three end points, the activation energy is about 140-152 kcal/mol. Therefore, heat may induce a common intracellular phenomenon involving protein denaturation or aggregation that is responsible for the damage observed by division delay, chromosomal aberrations, and cell-killing. There are great differences in division delay induced during the cell cycle by heat or radiation. Division delay is the same when cells are heated in the relatively heat-resistant G1 phase or the relatively heat-sensitive S phase, with about 15 min of delay for 1 min of heating at 43 degrees C. This contrasts with the increase in division delay observed when cells are irradiated in the relatively radioresistant S phase compared with the relatively radiosensitive G1 phase. Quantitatively, division delay for a treatment that reduces survival to about 0.1 is 15 or 25 h for heating in S or G1, respectively, compared with only 6 or 1.5 h for irradiation in S or G1, respectively.

Thermal Tolerance during S Phase for Cell Killing and Chromosomal Aberrations

Synchronous Chinese hamster ovary cells in early S phase were obtained by selecting mitotic cells, accumulating them at the G1/S border by incubating them in aphidicolin for 12 h, and then incubating them for 2 h after releasing them from the aphidicolin block. To determine if thermotolerance could be induced, the cells were heated at 43 degrees C for 20 min in early S phase, incubated for 160 min, and then heated a second time at 43 degrees C for different durations (30-100 min). For the control, nontolerant population, the cells in early S phase were incubated for 50 min and then heated once at 43 degrees C for different durations (20-60 min). Flow cytometric analysis indicated that the population receiving the second heat dose was in the same part of S phase as the population receiving the single heat dose. A comparison of the heat response for the two populations indicated that heating during early S phase induced thermotolerance for both cell killing and chromosomal aberrations; i.e., for 10% survival, which corresponded to 10% of the cells being cytologically normal, the thermal dose was twofold greater in the thermotolerant cells than in the control, nontolerant cells. Furthermore, this thermotolerance developed during S phase. These observations support the hypothesis that heating during S phase kills cells primarily by inducing chromosomal aberrations.

Chromosome site‐specific immunohistochemical detection of DNA adducts in N‐acetoxy‐2‐acetylaminofluorene‐exposed chinese hamster ovary cells

In these studies a polyclonal antiserum elicited against a carcinogen-DNA adduct was used to explore the localization of DNA adducts in metaphase chromosomes of cultured cells. Morphological visualization of the adduct N-(deoxyguanosin-8-yl)-2-aminofluorene (dG-C8-AF) in Chinese hamster ovary (CHO) cells exposed to the direct-acting carcinogen N-acetoxy-2-acetylaminofluorene (N-Ac-AAF) was accomplished by indirect immunofluorescence with an anti-G-C8-AF antiserum. At the same time the pattern of chromosomal DNA replication was determined by replicative incorporation of bromodeoxyuridine (BrdUrd) and chromosomal staining with anti-BrdUrd. Visualization of DNA in chromosomes was accomplished with Hoechst 33258 dye. When synchronized CHO cells were exposed to N-Ac-AAF for 0.5 h during early S phase, the chromosomal pattern of dG-C8-AF adduct formation was not random. Metaphase chromosome spreads from cells exposed to N-Ac-AAF in different experiments contained certain chromosome regions that had a consistently high adduct concentration. The regions of high DNA damage corresponded to the regions active in DNA synthesis when BrdUrd and the carcinogen were given simultaneously in early S phase. In addition, the patterns of high adduct concentration and replicative synthesis shifted when the carcinogen and BrdUrd were given simultaneously during late S phase. Thus, the stage of cell cycle in which adducts are induced is an important factor in the specific location of the highest concentrations of this type of DNA lesion.

Acrylamide sensitization of the heat response of the cytoskeleton and cytotoxicity in attaching and well-spread synchronous Chinese hamster ovary cells.

The vimentin intermediate filament (VIMF) network is more sensitive to heat-induced disruption than either the microtubule (MT) or microfilament (MF) cytoskeletal (CSK) arrays in G1 Chinese hamster ovary (CHO) cells (Coss and Wachsberger: Radiation Research, 1987). We therefore investigated the effect of the VIMF disruptive agent, acrylamide (Eckert: European Journal of Cell Biology 37:169-174, 1985), on the heat response of synchronous CHO cells. Cells, either in the process of spreading (G1 or S phase) or in the well-spread state (S phase), were exposed to a nontoxic concentration of 5 mM acrylamide, heated, and processed for immunofluorescence microscopy 30 min or 20 hr following the heat shock. Recovery from CSK disruption was related to cell survival. CHO cells, either in the process of spreading or in the well-spread state, were sensitized to heat-induced CSK disruption and cytotoxicity by acrylamide. Recovery from CSK disruption correlated with surviving fractions of cells treated in the G1 phase but not with surviving fractions of cells treated in the S phase and was independent of the degree of cell spreading. This correlation suggests that damage to CSK structures may contribute to the death of cells treated in G1 but not necessarily to the death of cells treated in S phase. The degree of acrylamide sensitization of heat-induced CSK disruption was greater for cells exposed to acrylamide prior to spreading than for well-spread cells. Furthermore, normal spreading of cells was prevented when they were plated into medium containing acrylamide, suggesting that acrylamide interferes with the initial stages of attachment and spreading of these cells. These observations are interpreted in relation to the possible role that VIMFs, together with cortical MFs, may play in mediating cell surface focal contacts in the initial stages of cell attachment and spreading.

Phospholipid turnover during cell-cycle traverse in synchronous Chinese-hamster ovary cells. Mitogenesis without phosphoinositide breakdown.

The turnover of phospholipids was investigated in quiescent serum-starved Chinese-hamster ovary (CHO-K1) cells stimulated to progress through the cell cycle by the addition of dialysed bovine serum. A variety of radiolabelling techniques were employed to study the rapid effects of serum on phospholipids and later events during G1 and S phases of the cell cycle. Pulse-labelling studies using [32P]Pi revealed that there was a stimulation of the synthesis rate of all phospholipids investigated during the initial few hours after serum addition. The greatest stimulation (20-fold) was observed in phosphatidylcholine, and the smallest in the polyphosphoinositides (PPIs). Mock stimulation with serum-free medium caused a similar increase in PPI turnover, but little or no effect on turnover of other phospholipids. This effect could be accounted for by a stimulation of the turnover of cellular ATP pools increasing [32P]ATP specific radioactivity. Late G1 and S phases were associated with a decrease in the rate of synthesis of all phospholipids. Phosphatidic acid was the only phospholipid whose labelling fell below that in mock-stimulated cells during the period of the cell cycle. Stimulation of serum-starved cells that had been prelabelled with myo-[2-3H]inositol caused no change in the amounts of inositol trisphosphate, but both serum-stimulated and mock-stimulated cells exhibited similar small decreases in both inositol bisphosphate and inositol monophosphate, of approx. 30% after 30 s. When cells were serum-stimulated in the presence of 10 mM-Li+, there was no increase in the size of the total inositol phosphate pool. We conclude that mitogenic stimulation and cell-cycle traverse cause profound and complex effects on phospholipid turnover in CHO-K1 cells, but there is no evidence for a role of inositol lipid turnover in the proliferative response to serum in this cell line.

An analysis of DNA replication in synchronized CHO cells treated with benzo[ a]pyrene diol epoxide

Synchronized Chinese hamster ovary (CHO) cells treated with (±)7 β,8 α-dihydroxy-9 α,10 α-epoxy-7,8,9,10-dihydrobenzo[ a]pyrene (BP diol epoxide I) were used to test the ‘block-gap’ model of replicative bypass repair in mammalian cells. One feature of the model is that carcinogenic or mutagenic DNA adducts act as blocks to the DNA replication fork on the leading strand. Using synchronized CHO cells, the rate of S phase progression by BrdUrd labeling of newly replicated DNA was measured. The rate of S phase progression was reduced by 22% and 42%, when the cells were treated at the G1/S boundary with 0.33 and 0.66 μM BP diol epoxide I, respectively. Using the pH step alkaline elution assay, it was found that the reduced rate of S phase progression was due to a delay in the appearance of multiple replicon size nascent DNA. This observation was consistent with the frequency of BP-DNA adducts present in the leading strand. A second feature of the ‘block-gap’ model is that the adduct-induced blockage on the lagging strand will produce gaps. It was determined by the use of high-resolution agarose gel electrophoresis, that the ligation of Okazaki size replication intermediates was blocked in a dose-dependent manner in BP diol epoxide I treated, synchronized CHO cells. These data are consistent with a block to the leading strand of DNA replication at DNA-carcinogen adducts. An inhibition of the ligation of Okazaki size fragments by BP diol epoxide I implies a block to replication of the DNA lagging strand leading to gap formation. The data presented here are, therefore, supportive of the ‘block-gap’ model of replicative bypass repair in carcinogen damaged mammalian cells.

Comparison of synchronized Chinese hamster ovary cells obtained by mitotic shake-off, hydroxyurea, aphidicolin, or methotrexate.

Synchronized cell populations are necessary to study many aspects of cell biology. We have developed a method to obtain highly synchronized Chinese hamster ovary cell populations in S phase or G2 phase by utilizing mitotic selection followed by incubation with either hydroxyurea, aphidicolin, or methotrexate for 12 h. Flow cytometry analysis shows that the coefficient of variation in the spread of the cell population in S phase is as low as 6%. Drug toxicity studies compare the effects of the various drugs on G1 and S phase cells. The use of aphidicolin or hydroxyurea results in the most highly synchronized cell populations, but methotrexate yields inadequate synchronization. These results demonstrate that both aphidicolin and hydroxyurea are useful drugs for obtaining highly synchronized cell populations after an initial synchrony in mitosis. Aphidicolin is perhaps the best choice because of less toxicity to S phase cells when used in low concentrations.

The in vitro genetic effects of fibrous erionite and crocidolite asbestos.

Epidemiologic evidence has recently identified an association between an endemic outbreak of pleural and peritoneal mesothelioma in the Urgup region of Turkey and exposure to zeolite fibres. This malignancy is usually associated with exposure to asbestos dusts whose mineralogical characteristics differ from those of zeolites. The present study further defines the in vitro biologic activity of erionite, a common zeolite fibre found in the Urgup region of Turkey. Both erionite and crocidolite asbestos fibres were clastogenic in synchronous Chinese hamster ovary (CHO) fibroblasts. Both fibres also altered CHO ploidy. Erionite, unlike crocidolite or Min-U-Sil quartz, caused a slight increase in sister chromatid exchanges in synchronous CHO cells. Neither erionite nor crocidolite was mutagenic in a human lymphoblastoid cell line. Erionite fibres thus produced in vitro cytogenic changes similar to those caused by asbestiform mineral dusts and, like asbestos fibres, did not induce mutations in human lymphoblastoid cells.

Effect of pH and Cell Cycle Progression on Development and Decay of Thermotolerance

Synchronous Chinese hamster ovary cells were heated in G1 and incubated at 37 degrees C at pH 6.75 or pH 7.4 before they were heated a second time. The magnitude and rate of development and decay of thermotolerance were greatly reduced at pH 6.75. This was also observed for asynchronous cells. Furthermore, the heat-induced delay in cell cycle progression was greatly enhanced at low pH and correlated with the reduced rate for development and decay of thermotolerance. However, studies with [3H]TdR to kill cells entering S phase showed that the decay of thermotolerance is relatively independent of the cell cycle. Therefore, low pH apparently slows many cell processes, including those associated with heat-induced cell cycle delay and the rate of development and decay of thermotolerance.

Survival and cell cycle kinetics responses of Chinese hamster ovary cells, and clones of human adenocarcinoma of the stomach and astrocytoma to diaziquone (AZQ) in vitro.

The anticancer agent 1,4-cyclohexadiene-1,4-dicarbonic acid, 2,5-bis(aziridinyl)-3,6-dioxo-, diethyl ester (AZQ) (NSC 182986) was studied in vitro to determine survival, cell cycle stage sensitivity, and cell cycle kinetics effects. One hour treatments with AZQ doses ranging from 1 micrograms/ml to 25 micrograms/ml revealed that human stomach tumor clones were most sensitive of three cell types studied to to the killing effects of AZQ; this sensitivity was followed in order by human astrocytomas and Chinese hamster ovary (CHO) cells. Depending on the AZQ dose, nondividing CHO cells were 10 to 180 times more sensitive than dividing CHO cells. Synchronized CHO cells were most sensitive to AZQ's killing effects when treated at the late S/G2 phase boundary, with the overall order of sensitivity being late S/G2, G2, mid-S, and G1 phase. Mitotic cells were neither killed by doses used in these studies, nor were they inhibited in their progression from mitosis into the G1 phase. Synchronized CHO cells treated in all other phases of the cell cycle were either blocked completely or delayed for up to 2 hours in their progression through the cell cycle. Flow microfluorometry (FMF) studies on exponentially growing CHO cells demonstrated that even at noncytotoxic doses (1 microgram/ml), AZQ caused very large, but reversible enrichments of cells in the S and G2 phases of the cell cycle. Since AZQ has already been shown to be effective against a variety of animal and human tumors (especially brain tumors) the data reported here may be useful in designing more effective treatment schedules and drug combination regimens.

Induction of sister-chromatid exchanges by DNA-damaging agents and 12- O-tetradecanoyl-phorbol-13-acetate (TPA) in synchronous Chinese hamster ovary (CHO) cells

Synchronous CHO cells were obtained by mitotic selection; synchrony was maintained up to the 5th cell cycle. The mitotic cells were seeded into T-25 flasks or P-60 plastic petri dishes, and cultured for 1 h at 37°C, then the cells were treated by X-ray, UV light, and mitomycin C. The cells were then cultured for 2 cell cycles with TPA and BrdUrd and sister-chromatid exchanges (SCE) analyzed by the FPG method. Following X-irradiation, the frequency of induced SCE increased linearly with dose reaching a maximum of 19.8 times the control frequency after 200 rad. With higher doses, the SCE frequency declined. In the presence of TPA, SCE frequencies were 1.8 times control levels for all X-ray doses studied (0–800 rad), the frequency seen in non-irradiated cultures treated with TPA. The induced SCE frequency also increased linearly following treatment with UVL and mitomycin C, reaching levels higher than 1.8 times controls with doses exceeding 2.5 J/m 2 UVL or mitomycin C (30 min). In the presence of TPA, the SCE frequencies increased to 1.8 times controls following low UVL and mitomycin C doses, but were not influenced by TPA in the higher dose range (above 2.5 J/m 2 or 10 −10 M mitomycin C. Most of the SCE were induced by X-rays during the first S phase after treatment. Following higher UVL doses (5 J/m 2), however, the SCE frequency remained elevated (1.5 times controls) for 4 cell cycles after exposure.

Repair capability and the cellular age response for killing and mutation induction after UV

The cell-cycle response for killing and mutation induction by ultraviolet irradiation was measured in synchronous Chinese hamster ovary cells (CHO wild-type) and in a UV-hypersensitive mutant (43-3B) derived from this line. The CHO 43-3B line shows a greatly enhanced sensitivity to killing ( D 0 of 0.3 as compared to 3.2 J/m 2 for the wild-type), is hypermutable, and deficient in DNA repair. For the wild-type, a characteristic age response is seen for killing by UV, with maximum sensitivity in early-S and resistance increasing through the S-phase. There is also a life-cycle specificity for induction of diphteria-toxin resistance in late-G 1 and early-S. Relatively little variation is seen through the cell cycle for induced 6-thioguanine and ouabain resistance. In contrast, the 43-3B cell line shows a relatively ‘flat’ response to UV throughout the cell cycle, for both killing and mutation induction. Therefore it appears that the characteristic age responses seen in the wild-type CHO are associated with the function of an essentially erro-free repair process. A variation in the ability of this repair process to act in eliminating potentially lethal and mutagenic lesions (either due to a variation in repair enzyme activities through the cell cycle, or in the time available for repair) would account for most of the age response which is seen in the wild-type for killing and mutation induction by ultraviolet light.

X-Ray Induction of 8-Azaguanine-Resistant Mutants in Synchronous Chinese Hamster Ovary Cells

Synchronous Chinese hamster ovary cells obtained by mitotic selection were x-irradiated at various points in the cell cycle and were analyzed for the frequency of mutants resistant to 8-azaguanine (AG). The induced frequencies were highest in mitosis (40 x 10/sup -5/ for 5 Gy), in early G/sub 1/ (30 x 10/sup -5/), and at the G/sub 1//S border (30 x 10/sup -5/); were low in mid- to late G/sub 1/ (15 x 10/sup -5/); and were lowest in S phase (6 x 10/sup -5/). Qualitatively, the variation in sensitivity during the cell cycle for induction of mutants corresponded with that reported for induction of chromosomal aberrations. However, a quantitative comparison of the two endpoints and plots of the logarithm of survival versus mutant frequencies or aberration frequencies indicates that the mutant frequencies were much higher than expected when the cells were irradiated in mitosis or at the G/sub 1//S border. A spectrum of mutants was observed and classified either as complete, i.e., failing to survive in azaserine-hypoxanthine medium and incorporating (/sup 3/H)hypoxanthine ((/sup 3/H)Hx) at a rate less than 1% of that for wild-type cells, or as partial, i.e., surviving in azaserine-hypoxanthine medium and incorporating (/sup 3/H)Hx at a ratemore » grater than 1% of that for wild-type cells. THe percentage of mutants classified as partials was 0 to 22% for selection in 30 ..mu..g/ml AG and 15 to 60% for selection in 5 ..mu..g/ml AG. However, the number of partial relative to complete mutants had no or little dependence on radiation dose or phase of the cell cycle irradiated. Seventeen mutants (10 partials and 7 completes) were cultured for 12 to 33 weeks in the absence of AG, and only one partial reverted to wild type as assayed by AG resistance or uptake of (/sup 3/H)Hx. Furthermore, the complete mutants and some of the partials were resistant to 6-thioguanine.« less

Ethylnitrosourea-induced mutagenesis in asynchronous and synchronous Chinese hamster ovary cells

The toxic and mutagenic activity of the alkylating carcinogen N-ethyl- N-nitrosourea (ENU) was studied in Chinese hamster ovary (CHO) cells. Cell killing and induction of 6-thioguanine-resistant (TG 4) and ouabain-resistant (OUA r) mutants were determined as a function of ENU dose and treatment time in asynchronous cell populations. A dose-dependent induction of mutants was observed. The mutation frequency did not increase with longer than 30-mn treatment times, implying that ENU breaks down rapidly in the cell. When synchronous populations of CHO cells obtained by mitotic detachment were treated with ENU at various times during the cell cycle, ENU-induced reproductive death was strongly dependent on the position in the cell cycle at the time of treatment, the time of highest sensitivity being the beginning of the S period. The pattern of mutation induction by ENU over the cell cycle was quite different from the pattern for cell killing. The induction of TG r mutants seemed to be independent of cell-cycle time. The induction of OUA r mutants was also independent of cell-cycle time after a low ENU dose; however, after a high ENU dose the frequency of OUA r mutants varied during the cell cycle, with a slight enhancement in G 1 and a decrease in the early S period. There was no sign of enhanced mutation induction at the growing point for the two genetic markers tested.

Acceleration of CHO cells into mitosis and reduction of X-ray-induced G2 delay by cordycepin

The mitotic cell selection technique was used to monitor the effect of cordycepin and/or 100 rad of X-rays on the entry of asynchronous or synchronous Chinese hamster ovary cells into mitosis. Continuous exposure of asynchronous cells to 5–50 μg/ml of cordycepin caused a rapid increase in the relative numbers of cells entering mitosis. In irradiated cells, cordycepin also reduced a 120-min mitotic delay by about 80 min and shifted the X-ray transition point about 10 min farther away from mitosis. Further studies showed that synchronous cells, treated continuously with 15 μg/ml of cordycepin starting at mid-to-late S phase, proceeded into mitosis approx. 40 min ahead of controls. This acceleration was associated with a 30-min lengthening of S phase and a reduction in the length of G2 from 80 to about 10 min. Furthermore, cordycepin reduced the 70-min mitotic delay observed for cells irradiated in S phase by 20 min. In contrast to the results for treatment at mid-S phase, continuous treatment during G2 of unirradiated synchronous cells with 15 μg/ml of cordycepin had little effect on accelerating cells into mitosis, yet did reduce by about 60 min the 170-min mitotic delay observed for cells irradiated in G2. Unirradiated synchronous cells treated with cordycepin starting before mid-S did not reach mitosis. Thus, there are the following transition points or intervals for cordycepin: for treatment prior to mid-S phase, cell cycle progression through S is blocked; for treatment between mid-S and late S, progression through S continues but progression through G2 is accelerated; and for treatment during G2, the rate of progression in accelerated only if the cells have been irradiated. These results are discussed in relation to the synthesis during late S and G2 of critical protein molecules essential for mitosis.

Effect of damage to early, middle, and late-replicating DNA on progress through the S period in Chinese hamster ovary cells

In order to explain sequential replication of DNA in eukaryotic cells, the duplication of a given bank of replicons is proposed to be initiated by specific events coupled to synthesis of the preceding replicons in the sequence. This model predicts that DNA synthesis in mid or late S should depend upon the synthesis and/or integrity of previously replicating DNA, but should not depend upon the integrity of DNA replicating later in the sequence. By incorporating BUdR into DNA during a given short interval of one S period in synchronous Chinese hamster ovary cells, we are able to selectively damage this DNA by irradiation at selected times before or during the next S period. Utilizing this technique, we find that damage to early replicating DNA before entry into the second S phase markedly suppresses DNA synthesis in the entire S period. Damage to mid or late replicating DNA prior to entry into the second S period has no effect on early S, but markedly reduces DNA synthesis commencing in mid or late S, respectively. Furthermore, if early replicating DNA is damaged with light in mid-S, no effect on subsequent DNA synthesis is observed. These results can be fitted to a model in which sequential triggering of replicon synthesis promotes orderly progression through S.

X-ray-induced mutants resistant to 8-azaguanine. II. Cell cycle dose response

Synchronous Chinese hamster ovary cells were irradiated in G 1 or S phase. Colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 μg/ml 8-azaguanine (AG). An expression period of over three generations (multiplicity of 20) was utilized, with expression times ranging from 58 to 114 h. Both G 1 and S phase were practically identtical in sensitivity to X-ray-induced mutations, with mutant frequency/viable cell/rad ranging from 1 × 10 −7 (75–100 rad) to 8 × 10 −7 (1000 rad). The spontaneous mutation rate, shown by Luria-Delbruck fluctuation analysis, was 5 × 10 −7 per generation. Thirty-three mutants, isolated at random and grown for over 30 generations in the absence of AG, were analyzed for plating efficiency ( PE) in different concentrations of AG or in hypoxanthine-aminopterin-thymidine (HAT) medium. Of these, 64% were resistant ( PE > 0.1) to 7.5 μg/ml AG, 85% to 5.0 μg/ml, and 91% to 3.5 μg/ml. Only 42% showed possible hypoxanthine-phosshoribosyltransferase (HPRTase) deficiency as evidenced by HAT sensitivity ( PE < 0.1). Wild type controls exhibited PE's)in 3.5 μg/ml AG of < 0.001 and in HAT of > 0.5. Of ten mutants studied, all demonstrated survival response to radiation similar to wild cells ( D̃ 0 of approx. 120 rad). For radiation protection standards, the radiation dose required to induce mutations at a rate equal to that occuring spontaneously is called the doubling dose. The doubling dose observed for acute irradiation was about 3 rad and was estimated to be 10–60 rad for chronic irradiation, similar to that often reported for in vivo studies.