DNA Synthetic Period Research Articles

X-Ray Sensitivity during the Cell Generation Cycle of Cultured Chinese Hamster Cells1

Our earlier reports (1–3) have provided a general, qualitative description of the X-ray sensitivity of Chinese hamster cells as a function of their age within the generation cycle. With a partially synchronized cell population and colony survival used as an assay, it was established that cells were least sensitive to X-rays in the latter part of the DNA synthetic period (S period) and appeared to be about equally sensitive in the pre-DNA synthetic period (G1) and post-DNA synthetic period (G2). The variations in sensitivity throughout the cycle seemed mainly but not exclusively due to variations in the threshold or shoulder of the survival curve. We were, however, aware of the fact that even a few resistant S cells present during stages we had designated as G1 or G2 could significantly alter the survival curve for G1 and G2 cells. Such S cells may be present not only because of inadequacies in the synchronization procedure but also because of differences in the rate of progression of cells through the cycle which tends to desynchronize the population. Even if the number of S cells present were known, their exact contribution to the survival curve would be difficult to estimate, especially if the sensitivity of S cells varied throughout the S period. To determine X-ray sensitivity more accurately at all stages, we have used lethal amounts of tritiated thymidine (HTdR) to inactivate the S cells. Furthermore, since the previous technique could not be used to obtain the sensitivity of mitotic (M) cells, we have modified our procedure so that this may be accomplished. Finally, in view of the possible association between X-ray resistance and DNA synthesis, it was important to find out whether cells in S had a constant or variable sensitivity throughout the synthetic period. MATERIALS AND METHODS

Effect of Irradiation on DNA Synthetic Period of the Mitotic Cycle in Cells

Kinetics of DNA synthesis in mitotic cycle of mouse corneal epithelial cells after single γ-irradiation (4 Gy) at the end of S period was studied by the method of radioautography. Normally, S period of corneal epithelial cells consists of several stages separated by intervals without DNA synthesis. The estimated mean duration of the first (S(1)) and second (S(2)) phases of S period was 16 and 10 h, respectively, and the interval between them was 7 h. Single irradiation at the end of S period changed the duration of mitotic cycle periods: S(2) phase became 2.2-longer than S(1) phase and the duration of g(1) period decreased, because the time for reparation in irradiated cell increases at the expense of g(1) period. Shortening of g(1) period is a factor promoting the appearance of transformed cells.

Effects of adenosine 5′-monophosphate on epidermal turnover

The structure and function of the epidermis is maintained by cell renewal based on epidermal turnover. Epidermal turnover is delayed by aging, and it is thought that the delay of the epidermal turnover is a cause of aging alternation of skin. The epidermal turnover is related to the energy metabolism of epidermal basal cells. Adenosine 5'-triphosphate (ATP) is needed for cell renewal: cell division, and adenosine 5'-monophosphate (AMP) increases the amount of intracellular ATP. These findings suggest that AMP accelerates the epidermal turnover delayed by aging. This study investigated whether AMP and adenosine 5'-monophosphate disodium salt (AMP2Na) accelerates the epidermal turnover. An effect of AMP2Na on cell proliferation was examined by our counting of keratinocytes. An effect of AMP2Na on cell cycle was examined by our counting of basal cells in DNA synthetic period of hairless rats. The effects of AMP2Na (or AMP) on the epidermal turnover were examined by our measuring stratum corneum transit time by use of guinea pigs, and by our measuring stratum corneum surface area by use of hairless rats and in a clinical pharmacological study. The AMP2Na showed two different profiles on the proliferation of primary cultured keratinocytes. At a low concentration it induced cell growth, whereas at a high concentration it inhibited cell growth. The number of basal cells in the DNA synthetic period of AMP2Na was significantly higher than that of the vehicle in hairless rats. The stratum corneum transit time of AMP2Na was significantly shorter than that of the vehicle in guinea pigs. The corneocyte surface area of emulsion containing AMP2Na was significantly smaller than that of the vehicle in volunteers. We conclude that AMP promotes the cell proliferation and the cell cycle progression of epidermal basal cells and accelerates epidermal turnover safely. In addition, AMP is useful for skin rejuvenation in dermatology and aesthetic dermatology.

Short life of stimulant released from ground skin slices.

SUMMARY Ground guinea-pig skin slices release a potent stimulant to epidermal cells which causes them to enter DNA synthesis (Hell, 1970). Further experiments have shown that this effect on the epidermis is immediate. If the stimulant-containing medium is replaced 15 min after setting up the experiment by fresh medium containing no stimulant, the epidermal cells have already received the impetus to enter the DNA synthetic period, and the original medium is without its characteristic effect on a further set of skin slices.

Differential Changes of CDK Activities in Glomeruli and Tubules during the Active DNA Synthetic Period after Ischemic Injury

The present study was designed to determine the spatial correlation among extent of DNA synthetic activity, expressions of G₁/S phase cyclins, cyclin-dependent kinases (CDKs) and CIP/KIP family of CDK inhibitors (CKIs), and activities of G₁/S phase CDKs in glomeruli and outer medullae of kidneys during the active regeneration period after ischemic injury. DNA synthetic activity was measured using [³H]-thymidine autoradiogram in the kidney sections. Cyclin, CDK, and CKI proteins were determined by Western blot analysis. CDK activities were determined by phosphorylation amount using specific substrate. The protein levels of cyclins (D1, D3, E, A) and activities of CDK4 and CDK2 were increased concomitant with the induction of DNA synthetic activity in outer medullae, but not in glomeruli, in adult kidneys during DNA synthetic period after ischemic injury. The p27^KIP1 protein, but not the p21^CIP1 protein, increased equally in total kidney, glomeruli, and outer medullae after ischemic injury. These results indicate that renal tubules have an active cyclin/CDK system, while glomeruli, do not have a cyclin/CDK system during active regeneration of kidneys after ischemic injury.

Improved development to blastocyst of ovine nuclear transfer embryos reconstructed during the presumptive S-phase of enucleated activated oocytes.

The timing of pronuclear formation and the initiation and duration of the DNA synthetic period (S-phase) were determined during the first cell cycle of electrically activated ovine oocytes matured in vivo. Reconstructed embryos were produced by electro-fusion-mediated nuclear transfer of unsynchronized single blastomeres. These were derived from embryos produced in vivo at the 16-cell stage (Day 4) and transferred to enucleated metaphase II oocytes at the time of activation or to enucleated activated oocytes during early, mid, and late stages of the presumptive S-phase. The frequency of development to blastocyst was greatest in embryos reconstructed during the presumptive S-phase of enucleated activated oocytes than in embryos reconstructed at the time of activation (mean 55.4% vs. 21.3%). No significant differences were observed when embryos were reconstructed during early, mid, or late stages of the presumptive S-phase (61.3%, 45.7%, and 57.7%, respectively). The results indicate that the use of enucleated activated oocytes as cytoplasts for embryo reconstruction can increase the frequency of development to blastocyst of embryos reconstructed from unsynchronized donor blastomeres.

Different effectiveness of 4-nitroquinoline-1-oxide, mitomycin C and ethyl methanesulfonate to induce lesions in DNA leading to sister chromatid exchange throughout successive cell cycles in Chinese hamster ovary cells.

The present study was carried out in Chinese hamster ovary cells in order to determine whether lesions induced by three different mutagens, namely 4-nitroquinoline-1-oxide (4-NQO), Mitomycin C (MMC) and Ethyl methanesulfonate (EMS), can persist for more than one cell generation leading to sister chromatid exchanges (SCE) or, alternatively, they are efficiently repaired during the next replicative period after treatment. In order to accurately score the number of SCEs arising during the first (S1), second (S2) and third (S3) DNA synthetic periods, third-cycle (M3) metaphases showing three-way differential (TWD) staining were analyzed. Our results show that, even though the three compounds tested were efficient in increasing the yield of SCE, the frequency of SCE was more dramatically increased after MMC treatment. Differences were also observed among the three mutagens with regard to the persistence of the lesions leading to SCE throughout successive cell generations. EMS-induced lesions appeared as more persistent than those induced by MMC. However, most of the damage induced by the UV mimetic agent 4-NQO seems to be efficiently repaired after the first round of DNA replication following treatment with the drug.

Developmental Timing in Fertilized Merogones of the Sand Dollar, Clypeaster japonicus: (merogone/cleavage/sea urchin/DNA synthesis/mitosis).

Pairs of nucleate and non-nucleate fragments prepared by manually bisecting unfertilized eggs of Clypeaster japonicus were inseminated to obtain a pair of egg fragments, one with a diploid nucleus (zygote) and the other with a male pronucleus (merogone). Haploid male pronuclei in the merogone always entered the first mitosis (NEB) later than the zygote partner. The delay in NEB ranged from 6 to 24 min in different pairs. The interval from the first NEB to formations of the karyomere and the cleavage furrow, and the durations of the second, third and fourth cell cycles were identical in the zygote-merogone pairs. Observations of Colcemid-treated eggs suggested that the duration of the first DNA synthetic period was prolonged in the merogone. From this observation of wide variation in the time of the first mitosis, in contrast to constant durations of subsequent mitotic intervals, we suppose that some critical event triggers the transition of development for the start of well-ordered sequences in the mitotic cycle in sea urchin development.

Cloning and cell cycle-dependent expression of DNA replication gene dnaC from Caulobacter crescentus

Chromosome replication in the asymmetrically dividing bacteria Caulobacter crescentus is discontinuous with the new, motile swarmer cell undergoing an obligatory presynthetic gap period (G1 period) of 60 min before the initiation of DNA synthesis and stalk formation. To examine the regulation of the cell division cycle at the molecular level, we have cloned the DNA chain elongation gene dnaC from a genomic DNA library constructed in cosmid vector pLAFR1-7. To ensure that the cloned sequence corresponded to dnaC, we isolated the gene by genetic complementation of the temperature-sensitive allele dnaC303 on DNA fragment that contained a Tn5 insertion element tightly linked by transduction to dnaC. The size of the dnaC gene was estimated to be 1,500 bp or less based on the pattern of complementation by subcloned restriction and BAL 31 deletion fragments. Nuclease S1 assays were used to map the transcription start site and to determine the pattern of dnaC expression in the cell cycle. Large amounts of the dnaC transcript began to accumulate only in the late G1 period of the swarmer cell and then peaked early during chromosome replication. We confirmed that the gene is periodically transcribed by monitoring the rate of beta-galactosidase synthesis directed by a dnaC promoter-lacZ fusion in a synchronous cell culture. dnaC is the first C. crescentus cell cycle gene whose regulation has been reported, and the discontinuous pattern of its expression suggests that the DNA synthetic period in these dimorphic bacteria is regulated in part by the stage-specific expression of DNA replication genes.

Effect of two aliphatic aldehydes, methylglyoxal and 4-hydroxypentenal, on the growth of Yoshida ascites hepatoma AH-130

The influence of a ketoaldehyde, methylglyoxal (MG), and a hydroxyalkenal, 4-hydroxypentenal (HPE), on the growth of a highly-deviated tumour has been investigated. MG and HPE, administered intraperitoneally, strongly depressed in rats the proliferative activity of the Yoshida ascites hepatoma AH-130, reducing its mitotic and labelling indices as well as the proportion of cycling cells (growth fraction). Monitoring the effects on the cell cycle by the labelled mitoses method showed that the percentage of labelled mitoses was markedly lowered after either aldehyde, which is indicative for a blocking effect in the S phase. In addition, the mean cell cycle time was slightly prolonged by MG, probably due to accumulation of cells in G1, whereas HPE delayed the first mitotic peak and increased the mean DNA synthetic period without modifying the overall cycle time. The effects of HPE on the cell cycle were prevented by pretreatment with polyamines. Repeated doses of MG significantly increased the fraction of tumour-bearing rats surviving at 90 days (‘indefinite’ survivors) as well as the survival time of those which succumbed, implying that the carcinostatic effect of MG persisted over several cell cycles. By contrast, HPE did not significantly modify the survival of AH-130-bearing rats, suggesting that its influence on tumour growth was rapidly reversible.

The Cell Cycle and Cell Population Kinetics in the Programmed Cell Death in the Limb-Buds of Normal and 5-Bromodeoxyuridine- Treated Chick Embryos: (cell death/BrdU/cell cycle/limb-bud).

The cell cycle and cell population kinetics have been analyzed in the interdigital regions of chick limb-buds during the course of programmed cell death both in normal and the 5-bromodeoxyuridine (BrdU)-treated embryos. Our previous study has shown that a single administration of BrdU at day 6 1/3 inhibited the programmed cell death occurring in normal development of limb-buds. Pulse- as well as continuous labelings with tritiated thymidine (3 H-TdR) were used. The results obtained from the analyses made on both normal and experimental embryos have demonstrated the presence of a particular DNA-synthetic period, around day 6 1/3, closely related to the programmed death occurring on day 7 1/3. In normal embryos, new cell populations, which did not belong to any phases of normal cell cycle, made their appearances in the process of programmed cell death. A possible correlation between programmed cell death and the cell cycle has been discussed in relation to the morphogenesis of limbs in both normal and BrdU-treated embryos.

Possible role of heterochromatin in chromosome breakage induced by tissue culture in oats (Avena sativa L.)

Perturbations in the replication of heterochromatic regions during tissue culture may be the cause of chromosome alterations observed in regenerated hexaploid oat plants (Avena sativa L.). An idiogram based on pachytene chromosome analysis was developed depicting the heterochromatin distribution on 17 of the 21 chromosomes. Heterochromatic regions were present pericentromerically on each chromosome, at telomeres in five of the chromosomes, and in the NORs of the three satellited chromosomes. Centromeric heterochromatin covered most of the short arm in several chromosomes. Tritiated-thymidine autoradiography was used to determine the nuclear cycle duration (G1 + S + G2 + mitosis = 0.8 + 5.2 + 0.3 + 3.8h = 10.1 h) in root-tip cells and revealed regions of late replication around the primary and secondary constrictions and at chromosome ends. Replication of heterochromatic regions late in the DNA synthetic period makes it plausible that these regions are more sensitive to nuclear cycle alterations caused by the culture process. Pachytene analysis of a heteromorphic chromosome pair that originated during culture showed the break in the deficient chromosome to be in centromeric heterochromatin. The presence of proximal and telomeric heterochromatin means that bridges resulting from replication problems in these regions could result in various alterations including deficiencies, duplications, and translocations and can account for heterochromatin amplification, all of which have been observed in plants that have been regenerated from tissue culture. Key words: heterochromatin, meiosis, telocentric chromosomes, oats, tissue culture, variation.

Temporal order of replication of genes responsible for hypoxanthine phosphoribosyl transferase and Na+/K+ ATPase in chemically transformed human fibroblasts.

The cytotoxic and mutagenic effects of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of a chemically induced, transformed line (Hut-11A cells) derived from diploid human skin fibroblasts were examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-h periods during the S phase with 5-bromodeoxyuridine (BrdU) followed by irradiation with near-ultraviolet (UV). The BrdU-plus-irradiation treatment was cytotoxic and mutagenic, while treatment with BrdU alone or irradiation alone was neither cytotoxic nor mutagenic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 h of S phase, whereas scare lethality was observed in late S phase. The BrdU-plus-irradiation treatment induced ouabain- and 6-thioguanine-resistant mutants, while BrdU alone or irradiation alone was not mutagenic. Ouabain-resistant mutants were induced during early S phase by the BrdU-plus-irradiation treatment. 6-Thioguanine-resistant mutants, however, were induced during middle to late S phase. These results suggest that a certain region or regions in the DNA of Hut-11A cells, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by BrdU treatment plus near-UV irradiation for cell survival and that gene(s) responsible for Na+/K+ ATPase is replicated during early S phase and gene(s) for hypoxanthine phosphoribosyl transferase is replicated during middle to late S phase.

Presence of an Allocyclic Early Replicating X Chromosome in Female Embryos of the Rat, Chinese Hamster and Rabbit: (X chromosome inactivation/allocyclic X chromosome/rat/Chinese hamster/rabbit).

Previous studies on early female mouse embryos revealed the presence of two kinds of inactive X chromosomes, one replicating late and the other early in the DNA synthetic period. The X chromosome that replicates early is of special interest because of its paternal origin, preferential occurrence in trophectoderm and primitive endoderm derivatives, and programmed shift to the late replicator. This study by BrdU labeling and acridine orange fluorescence staining was undertaken to examine whether the inactive X chromosome behaves in a similar manner in other laboratory mammals. In rat embryos the paternal X chromosome was found to show the same behavior in extraembryonic tissues. Early replicating chromosomes were also found in the extraembryonic regions of Chinese hamster and rabbit embryos, although their parental origin could not be determined due to the absent of X chromosome polymorphism in these species. Probably the early replicating X chromosome occurs commonly in mammals. Its functional significance is unknown.

Dependence of lethality induced by a direct DNA perturbation of synchronized human diploid fibroblasts on different periods of the DNA synthetic period (S phase).

The cytotoxic effect of a direct perturbation of DNA during various portions of the DNA synthetic period (S phase) of cultured human diploid fibroblasts was examined. The cells were synchronized by a period of growth in low serum with a subsequent blockage of the cells at the G1/S boundary by hydroxyurea. This method resulted in over 90% synchrony, although approximately 20% of the cells were noncycling. Synchronized cells were treated for each of four 2-hour periods during the S phase with 5-bromodeoxyuridine (0.1-10 microM), followed by irradiation with near-UV (5-10 min). The 5-bromodeoxyuridine-plus-irradiation treatment was cytotoxic, while treatment with 5-bromodeoxyuridine alone or irradiation alone was not cytotoxic. The cytotoxicity was dependent upon the periods of S phase during which treatment was administered. The highest lethality was observed for treatment in early to middle S phase, particularly in the first 2 hours of S phase, whereas scarce lethality was observed in late S phase. The extent of substitution of 5-bromodeoxyuridine for thymidine in newly synthesized DNA was similar in every period of the S phase. Furthermore, no specific period during S phase was significantly more sensitive to treatment with respect to DNA damage, as determined by an induction of unscheduled DNA synthesis. These results suggest that a certain region or regions in the DNA of human diploid fibroblasts, as designated by their specific temporal relationship in the S phase, may be more sensitive to the DNA perturbation by 5-bromodeoxyuridine treatment plus near-UV irradiation for cell survival.

Delay In Entry Into S Phase After Heat Shock

Synchronized cells of the Harding Passey melanoma grown in culture were given a heat shock treatment of 44 degrees C for 36 min. Thymidine incorporation was measured at frequent intervals after heat shock to determine the time of onset of the next DNA synthetic period. If the heat shock was given at the end of G1, the following S was delayed by 20 hr. Heating at other times in the cell cycle resulted in an even longer interval before the onset of S. The end of G1 was also the most resistant to hyperthermic killing and to the effect of heat on the magnitude of thymidine incorporation in the following S. Heating the cells a second time did not repeat the effect of the first treatment unless the second heat shock treatment was at a considerably higher temperature. Thus thermotolerance to heat shock killing also applies to cell-cycle delay.

Relationships between DNA synthesis and mitotic events in fertilized sea urchin eggs: Aphidicolin inhibits DNA synthesis, nuclear breakdown and proliferation of microtubule organizing centers, but not cycles of microtubule assembly

The synthesis of DNA in fertilized eggs of the American Gulf Coast sea urchin Lytechinus variegatus is 90% inhibited in the presence of 5.0 μg/ml aphidicolin. This inhibition may be imposed immediately upon addition of aphidicolin to the external medium when embryos are in “S” phase. Observations of living embryos with Nomarski optics and time-lapse video microscopy reveal that when eggs are fertilized and cultured in the continuous presence of aphidicolin, nuclear envelope breakdown, chromosome condensation, and cytokinesis are inhibited. All other post-fertilization events observable with this technique, including the assembly and disassembly of a bipolar spindle, proceed in the presence of aphidicolin. Antitubulin immunofluorescence microscopy of aphidicolin-arrested embryos demonstrates that microtubules attempt to assemble a mitotic apparatus at the first cell cycle; the arrested intact zygote nucleus is embedded within this bipolar structure. Subsequent cycles of microtubule assembly and disassembly proceed roughly on schedule with later division cycles, but the microtubule organizing centers (MTOC's) are unable to duplicate properly and irregular monasters are observed. If aphidicolin is added to embryos after the first DNA synthetic period, nuclear envelope breakdown, chromosome condensation, and cytokinesis proceed for that cycle and the embryos arrest at the two-cell stage. These results suggest that the direct inhibitory effects of aphidicolin may well be limited to the synthesis of DNA, which itself regulates nuclear cycles independently from the subsequent generation of mitotic poles, and that cytoplasmic clocks regulate microtubule assembly cycles but not the configuration of microtubule arrays.

Effects of some systemic fungicides on the chiasma frequencies in Hordeum vulgare.

Four systemic fungicides-thiabendazole, fuberidazole, carboxin and oxycarboxin have been tested for effects on chiasmata in barley. All compounds excepting thiabendazole have depressed chiasma frequencies significantly. The source of such a variation could be laid a) fairly very early in development as the treatment was done during the first DNA synthetic period in the seeds or b) much later during development because the compounds are systemic in nature. The observations are likely to be useful in genetic toxicology and in dealing with experimental crop populations.

Induction of sister chromatic exchanges and inhibition of cellular proliferation in vitro. I. Caffeine.

Cytogenetic assay systems based on the detection of sister chromatid exchanges (SCE) are widely advocated as a sensitive screening method for assessing genotoxic potential. While many agents have been examined for their ability to induce SCE's, complete dose-response information has often been lacking. We have reexamined the ability of one such compound-caffeine-to induce SCEs and also to inhibit cellular proliferation in human peripheral lymphocytes in vitro. An acute exposure to caffeine prior to the DNA synthetic period did not affect either SCE frequency or the rate of cellular proliferation. Chronic exposure to caffeine throughout the culture period lead to both a dose-dependent increase in SCEs (SCEd or doubling dose = 2.4 mM; SCE10 or the dose capable of inducing 10 SCE = 1.4 mM) and a dose-dependent inhibition of cellular proliferation (IC50 or the 50% inhibition concentration = 2.6 mM). The relative proportion of first generation metaphase cels, an assessment of proliferative inhibition, increased linearly with increasing caffeine concentrations. However, SCE frequency increased nonlinearly over the same range of caffeine concentrations. Examination of the ratio of nonsymmetrical to symmetrical SCEs in third generation metaphase cells indicated that caffeine induced SCEs in equal frequency in each of three successive generations. The dependency of SCE induction and cellular proliferative inhibition on caffeine's presence during the DNa synthetic period suggests that caffeine may act as an antimetabolite in normal human cells. The significance of these results in regard to both caffeine's genotoxic potential and to the reliability of the SCE assay system are discussed.

Increased cell renewal of epithelial cells in the gallbladder induced by lithogenic diet (author's transl)

Mice fed a diet containing 1% cholesterol and 0.5% cholic acid develop gallstones within six or eight weeks. Experimental lithogenesis initiates an increase in the proliferative activity of epithelial cells in the gallbladder well before the appearance of gallstones. During the initial stages of experimental cholelithiasis an increase in the number of labelled (tritiated thymidine) nuclei in mitosis and in the DNA-synthetic period was found. The labelled nuclei were observed particularly on the epithelial surface of the gallbladder; many of them were about to exfoliate into the lumen. These data were confirmed by applying vincristine. We would suggest that the rapid exfoliation of the proliferating cells could be one of the nucleating factors that promote gallstones formation.