DNA synthesis and cell cycle progression of synchronized L-cells after irradiation in various phases of the cell cycle

Summary The varying sensitivity to radiation in the different phases of the cell cycle was investigated using L-929 cells of the mouse. The cells were synchronized by mechanical selection of mitotic cells. The synchronous populations were X-irradiated with a single dose of 10 Gy in the middle of the G₁-phase, at the G₁/S-transition or in the middle of the S-phase, respectively. The radiation effect was determined in 2 h intervals a) by¹⁴C-TdR incorporation (IT) into the DNA, b) by autoradiography (AR), c) by flow cytometry (FCM). The incorporation rate decreased in all three cases, but the reasons appeared to be different, as can be derived from FCM and AR data: After irradiation in G₁, a fraction of cells was prevented from entering S-phase, after irradiation at G₁/S a proportion of cells was blocked in the S-phase, and after irradiation in S, DNA synthesis rate was reduced. As a consequence of these effects, the mean transition time through S-phase increased. The G₂ blocks, obtained after irradiation at the three stages of the cycle were also different: Cells irradiated in G₁ are partly released from the block after 10 h. Irradiation at G₁/S caused a persisting accumulation of 50% of the cells in G₂, and for irradiation in S more than 80% of the cells were arrested in G₂.     

DNA repair in UV-irradiated heteroploid cells at different phases of the cell cycle

The variation of DNA repair activity during the cell cycle was studied by analysing the UV-stimulated DNA synthesis in cells synchronized in mitosis. This activity was detected both by autoradiography and by directly measuring the incorporation of tritiated thymidine in cells irradiated and incubated in the presence of hydroxyurea. Cells in all phases were found to be able to perform repair. However the activity appeared to be considerably lower in mitotic cells than in cell in other phases. Increasing values of repair capacity were observed in G1 cells, in mixed G2, S and M cells and in asynchronous cells. The relationship between these findings and data on survival rates in the same synchronized cells is discussed.     

A new method for the estimation of cell cycle phases.

A method is described, which is applicable to cell renewal systems with an anatomical structure in which all cell locations may be uniquely mapped. Its use is demonstrated on the rat incisor inner enamel epithelium, which forms a one cell thick column in the sagittally sectioned tooth. Cells born in the apical part of the column migrate toward the distal end of the tooth, where they mature. As the cells migrate along the column, they traverse the various cell cycle phases. The present study has been designed to estimate the probability of a cell being in a given phase; all cells touching the basement membrane were numbered, and the number of cells separating any two cells was taken as a measure of distance. Since generally all cells move in one direction (lateral cell migration may occur), it is possible to solve the problem with the aid of functions describing the renewal counting stochastic process in which cell distance serves as an independent variable. The method predicts labelled cell and mitotic rates which agree with those estimated in the usual way. It was then utilized to estimate the fraction of cells in G2.     

Fine structure of DNA melting curves.

Theoretical calculations predict that the differential melting curves for random polynucleotide sequences having lengths up to several tens of thousands of base pairs have a clear-cut fine structure. This structure appears in the form of multiple narrow peaks 0.3–0.4°C wide on the bell shaped main curve. The differential melting curves have different shapes for different specific sequences. The theory also predicts the disappearance of the fine structure when the length of the sequence increases and when circular, covalently closed DNA is considered instead of the open structure. The predictions of the theory were confirmed by the measurements of differential melting curves for open and covalently closed circular forms of DNA for PM2 phage (N = 10⁴ base pairs) and also for other phage DNA's of different length: T7 (N = 3.8 × 10⁴); S_D (N = 9.2 × 10⁴); T2 (N = 17 × 10⁴). It was shown that the effect of fine structure results mainly from the cooperative melting out of DNA regions 300–500 base pairs long.     

Gene-specific and strand-specific DNA repair in the G1 and G2 phases of the cell cycle.

We have analyzed the fine structure of DNA repair in Chinese hamster ovary (CHO) cells within the G1 and G2 phases of the cell cycle. Repair of inactive regions of the genome has been suggested to increase in the G2 phase of the cell cycle compared with other phases. However, detailed studies of DNA repair in the G2 phase of the cell cycle have been hampered by technical limitations. We have used a novel synchronization protocol (D. K. Orren, L. N. Petersen, and V. A. Bohr, Mol. Cell. Biol. 15:3722-3730, 1995) which permitted detailed studies of the fine structure of DNA repair in G2. CHO cells were synchronized and UV irradiated in G1 or early G2. The rate and extent of removal of cyclobutane pyrimidine dimers from an inactive region of the genome and from both strands of the actively transcribed dihydrofolate reductase (DHFR) gene were examined within each phase. The repair of the transcribed strand of the DHFR gene was efficient in both G1 and G2, with no major differences between the two cell cycle phases. Neither the nontranscribed strand of the DHFR gene nor an inactive region of the genome was repaired in G1 or G2. CHO cells irradiated early in G2 were more resistant to UV irradiation than cells irradiated in late G1. Since we found no major difference in repair rates in G1 and G2, we suggest that G2 resistance can be attributed to the increased time (G2 and G1) available for repair before cells commit to DNA synthesis.     

The cell cycle phases of DNA damage and repair initiated by topoisomerase II-targeting chemotherapeutic drugs

Although cytostasis and cytotoxicity induced by cancer chemotherapy drugs targeting topoisomerase II (topoII) arise in specific cell cycle phases, it is unknown whether the drug-initiated DNA damage triggering these responses, or the repair (reversal) of this damage, differs between cell cycle phases or between drug classes. Accordingly, we used a flow cytometric alkaline unwinding assay to measure DNA damage (strand breakage (SB)) and SB repair in each cell cycle compartment of human cancer cell lines treated with clinically relevant concentrations of doxorubicin, daunomycin, etoposide, and mitoxantrone. We found that treated HeLa and A549 cells exhibited the greatest SB in G2/M phase, the least in G1 phase, and generally an intermediate amount in S phase. The cell cycle phase specificity of the DNA damage appeared to be predictive of the cell cycle phase of growth arrest. Furthermore, it appeared to be dependent on topoIIalpha expression as the extent of SB did not differ between cell cycle compartments in topoIIalpha-diminished A549(VP)28 cells. HeLa cells were apparently unable to repair doxorubicin-initiated SB. The rate of repair of etoposide-initiated SB in HeLa cells and of mitoxantrone-initiated SB in HeLa and A549 cells was similar in each cell cycle compartment. In A549 cells, the rate of repair of doxorubicin and etoposide-initiated SB differed between cell cycle phases. Overall, these results indicate that the cell cycle phase specificity of cytostasis and cytotoxicity induced in tumor cells by topoII-targeting drugs may be directly related to the cell cycle phase specificity of the drug-initiated DNA damage. Analysis by cell cycle compartment appears to clarify some of the intercellular heterogeneity in the extent of drug-initiated DNA damage and cytotoxicity previously observed in cancer cells analyzed as a single population; this approach might be useful in resolving inconsistent results reported in investigations of tumor cell topoII content versus response to topoII-targeting drugs.     

DNA damage bypass operates in the S and G2 phases of the cell cycle and exhibits differential mutagenicity

Translesion DNA synthesis (TLS) employs low-fidelity DNA polymerases to bypass replication-blocking lesions, and being associated with chromosomal replication was presumed to occur in the S phase of the cell cycle. Using immunostaining with anti-replication protein A antibodies, we show that in UV-irradiated mammalian cells, chromosomal single-stranded gaps formed in S phase during replication persist into the G2 phase of the cell cycle, where their repair is completed depending on DNA polymerase ζ and Rev1. Analysis of TLS using a high-resolution gapped-plasmid assay system in cell populations enriched by centrifugal elutriation for specific cell cycle phases showed that TLS operates both in S and G2. Moreover, the mutagenic specificity of TLS in G2 was different from S, and in some cases overall mutation frequency was higher. These results suggest that TLS repair of single-stranded gaps caused by DNA lesions can lag behind chromosomal replication, is separable from it, and occurs both in the S and G2 phases of the cell cycle. Such a mechanism may function to maintain efficient replication, which can progress despite the presence of DNA lesions, with TLS lagging behind and patching regions of discontinuity.     

DNA methylation profiling distinguishes histological subtypes of renal cell carcinoma

Renal cell carcinoma (RCC) accounts for around 3% of cancers in the UK, and both incidence and mortality are increasing with the aging population. RCC can be divided into several subtypes: conventional RCC (the most common, comprising 75% of all cases), papillary RCC (15%) and chromophobe RCC (5%). Renal oncocytoma is a benign tumor and accounts for 5% of RCC. Cancer and epigenetics are closely associated, with DNA hypermethylation being widely accepted as a feature of many cancers. In this study the DNA methylation profiles of chromophobe RCC and renal oncocytomas were investigated by utilizing the Infinium HumanMethylation450 BeadChips. Cancer-specific hypermethylation was identified in 9.4% and 5.2% of loci in chromophobe RCC and renal oncocytoma samples, respectively, while the majority of the genome was hypomethylated. Thirty (hypermethylated) and 41 (hypomethylated) genes were identified as differentially methylated between chromophobe RCC and renal oncocytomas (p < 0.05). Pathway analysis identified some of the differentially hypermethylated genes to be involved in Wnt (EN2), MAPK (CACNG7) and TGFβ (AMH) signaling, Hippo pathway (NPHP4), and cell death and apoptosis (SPG20, NKX6-2, PAX3 and BAG2). In addition, we analyzed ccRCC and papillary RCC data available from The Cancer Genome Atlas portal to identify differentially methylated loci in chromophobe RCC and renal oncocytoma in relation to the other histological subtypes, providing insight into the pathology of RCC subtypes and classification of renal tumors.     

Cytoplasmic phases in the first cell cycle of the activated frog egg

The first cell cycle of the activated frog egg is longer than subsequent cycles and several developmentally important events such as the determination of bilateral symmetry occur at this time. When eggs of Rana pipiens or Xenopus laevis are dissected at times after activation, differences in the consistency of the animal half cytoplasm can be detected visually, and the first cell cycle has been divided into four cytoplasmic phases on this basis. Phase 1 includes the events of activation and lasts about one-third of the first cycle. In phase 2, the cytoplasm becomes fluid except for the rigid, growing sperm aster, and most of the migration of the pronuclei occurs in phase 2. In phase 3, the cytoplasm becomes firm whether or not a sperm aster had been present, and the grey crescent forms, indicating the plane of bilateral symmetry. The firmness of the cytoplasm is colchicine sensitive but cytochalasin B insensitive as is grey crescent formation. In phase 4, the cortex detaches from the firm cytoplasm, and the firmness is now cytochalasin B sensitive and colchicine insensitive. The changes in cytoplasmic consistency during the first cell cycle probably reflect changes in the cytoskeleton, and the cytoplasmic consistency is functionally correlated with developmental events in the first cell cycle.     

A quantitative assay for the monitoring of autophagosome accumulation in different phases of the cell cycle

Macroautophagy is a process accompanied by the formation of double-membrane vesicles known as autophagosomes. Although in recently published reviews various methods for the detection of autophagosomes were described, a reliable technique for the automated quantitative evaluation of autophagosome accumulation is still lacking. Here we developed a new assay which is based on the fact that the number of autophagosomes is correlated with the amount of the LC3-II protein, which is specifically associated with autophagosomal membranes. Monitoring of autophagosome: accumulation was performed by extracting the membrane-unbound LC3-I form of the protein from cells, followed by flow cytometric detection of the autophagosomal membrane-associated fraction of LC3-II. This assay could be used for monitoring autophagosomes by flow cytometry utilizing immunostaining with the antibody against the LC3 protein. It is also suitable for analysis of: cells expressing GFP-LC3. We showed that co-staining with propidium iodide allows detection of basal level of autophagosomes in different phases of the cell cycle. Autophagy activators, such as: rapamycin or cell starvation, were able to induce accumulation of autophagosomes in G0/G1, S and G2/M phases. Thus, utilization of this assay simplifies monitoring of autophagosome accumulation induced by different activators or inhibitors of macroautophagy and it is suggested as being useful in the detection of autophagosomes in different phases of the cell cycle.     

Polysomes in various phases of the cell cycle in synchronized plasmacytoma cells

The fraction of membrane-bound and free polysomes during different phases of the cell cycle was determined in suspension cultures of mouse plasmacytoma cells, synchronized by growth in isoleucine-deficient medium. The membrane-bound polysomes reached a maximum value (about 28 % of total polysomes) during the G 1 phase. In the S phase and G 2 phase only 18 to 20 % of the total polysomes were found to be membrane-bound. A high percentage of membrane-bound polysomes in the G 1 phase of the cell cycle agrees with the earlier finding that maximum synthesis of immunoglobulin light chain takes place on polysomes bound to the membrane in the G 1 phase of the cell cycle. The presence of a significant fraction of membrane-bound polysomes in the S and G 2 phases of the cell cycle would suggest that membrane-bound polysomes are also involved in the synthesis of proteins other than immunoglobulins.The ultrastructure of the cells during the various phases of the cell cycle was also studied. During the G 1 phase the surface of the majority of cells was distinguished by the presence of ruffles and slender villus-like cytoplasmic projections. In the S phase the surface contour tended to become smooth and even. These differences in the surface morphology may reflect the change in function which occurs during the transition from the G 1 to the S phase.     

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells

The object of this study was to determine the kinetics of chromosome decondensation during the G₁ period of the HeLa cell cycle. HeLa cells synchronized in the G₁ period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N₂O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G₁ period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G₁ period, when DNA synthesis is initiated.     

Chromatin structure during the prereplicative phases in the life cycle of mammalian cells

The object of this study was to determine the kinetics of chromosome decondensation during the G1 period of the HeLa cell cycle. HeLa cells synchronized in the G1 period following the reversal of mitotic block were fused with Colcemid-arrested mitotic HeLa cells at 1.5, 3, 5, and 7 h after the reversal of N2O block. The resulting prematurely condensed chromosomes (PCC) were classified into six categories depending on the degree of their condensation. The frequency of occurrence of each category was plotted as a function of time after mitosis. The results of this study indicate that the process of chromosome decondensation, initiated during the telophase of mitosis continues throughout the G1 period without any interruption, thus the chromatin reaches an ultimate state of decondensation by the end of G1 period, when DNA synthesis is initiated.     

Base excision repair of ionizing radiation-induced DNA damage in G1 and G2 cell cycle phases

Background

Major genomic surveillance mechanisms regulated in response to DNA damage exist at the G₁/S and G₂/M checkpoints. It is presumed that these delays provide time for the repair of damaged DNA. Cells have developed multiple DNA repair pathways to protect themselves from different types of DNA damage. Oxidative DNA damage is processed by the base excision repair (BER) pathway. Little is known about the BER of ionizing radiation-induced DNA damage and putative heterogeneity of BER in the cell cycle context. We measured the activities of three BER enzymes throughout the cell cycle to investigate the cell cycle-specific repair of ionizing radiation-induced DNA damage. We further examined BER activities in G2 arrested human cells after exposure to ionizing radiation.

Results

Using an in vitro incision assay involving radiolabeled oligonucleotides with specific DNA lesions, we examined the activities of several BER enzymes in the whole cell extracts prepared from synchronized human HeLa cells irradiated in G1 and G2 phase of the cell cycle. The activities of human endonuclease III (hNTH1), a glycosylase/lyase that removes several damaged bases from DNA including dihydrouracil (DHU), 8-oxoguanine-DNA glycosylase (hOGG1) that recognizes 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) lesion and apurinic/apyrimidinic endonuclease (hAPE1) that acts on abasic sites including synthetic analog furan were examined.

Conclusion

Overall the repair activities of hNTH1 and hAPE1 were higher in the G1 compared to G2 phase of the cell cycle. The percent cleavages of oligonucleotide substrate with furan were greater than substrate with DHU in both G1 and G2 phases. The irradiation of cells enhanced the cleavage of substrates with furan and DHU only in G1 phase. The activity of hOGG1 was much lower and did not vary within the cell cycle. These results demonstrate the cell cycle phase dependence on the BER of ionizing radiation-induced DNA damage. Interestingly no evidence of enhanced BER activities was found in irradiated cells arrested in G2 phase.     

An insight into the structure of DNA through melting studies

Helix-coil transition of calf thymus (CT) DNA in 0.1 M NaCl was observed under different environmental conditions for studying structural and conformational changes, if any. Pre-exposure of the DNA to different types of radiations - UV and gamma-brought different degrees of change (lowering) in the melting temperature (Tm). Interaction of drugs (daunomycin and actinomycin-D) increased the Tm inducing structural stability. There was 50% more destabilisation in case of polynucleotide-drug complex as compared to DNA-drug complex exposed to UV radiation. Thermodynamic studies on DNA in D2O (0.1 M NaCl prepared in D2O) were carried out and compared with the corresponding results in H2O. Presence of D2O increased the stability of the DNA structure. Structural and conformational aspects have been discussed in the light of the results obtained.     

Variation in cell-substratum adhesion in relation to cell cycle phases

The quantification of focal adhesion sites offers an assessable method of measuring cell-substrate adhesion. Such measurement can be hindered by intra-sample variation that may be cell cycle derived. A combination of autoradiography and immunolabelling techniques, for scanning electron microscopy (SEM), were utilised simultaneously to identify both S-phase cells and their focal adhesion sites. Electron-energy 'sectioning' of the sample, by varying the accelerating voltage of the electron beam, combined with backscattered electron (BSE) imaging, allowed for S-phase cell identification in one energy 'plane' image and quantitation of immunogold label in another. As a result, it was possible simultaneously to identify S-phase cells and their immunogold-labelled focal adhesions sites on the same cell. The focal adhesion densities were calculated both for identified S-phase cells and the remaining non-S-phase cells present. The results indicated that the cell cycle phase was a significant factor in determining the density of focal adhesions, with non-S-phase cells showing a larger adhesion density than S-phase cells. Focal adhesion morphology was also seen to correspond to cell cycle phase; with 'dot' adhesions being more prevalent on smaller non-S-phase and the mature 'dash' type on larger S-phase cells. This study demonstrated that when quantitation of focal adhesion sites is required, it is necessary to consider the influence of cell cycle phases on any data collected.     

The induction of DNA strand breaks at specific sites by N-nitroso-N-ethylurea depends on the phases of the cell cycle

Synchronized root meristems of Pisum sativum were treated at each phase of the cell cycle with 6.25 mM N-nitroso-N-ethylurea. DNA extracted from treated cells and run in agarose gel electrophoresis exhibits a series of discrete fragments with length below 2500 bp and a significant number of unspecific single-stranded breaks (or alkali-labile sites). Experiments with micrococcal nuclease indicated that the nucleosomal organization of the chromatin is not responsible for the generation of the discrete fragments: it seems that their appearance is associated with a preferable attack of the mutagen at specific sites, characteristics for the plant genome. Moreover, a cell cycle dependent release of the discrete fragments was found with maximum at G1-S and minimum at mitosis. The model experiments designed to clarify this observation suggest that it might be determined from the cell cycle dependent fluctuation in the accessibility of the chromatin DNA and/or the process of excision-repair.