Prothymosin alpha expression is associated to cell division in rat testis

Using immunohistochemical methods, we have investigated the cellular distribution of prothymosin alpha (ProT) in adult rat testis. A policlonal antibody raised against thymosin alpha 1 conjugated to keyhole limpet hemocyanin was used. ProT immunoreactivity was observed in the cytoplasm and nucleus of spermatogonia and primary spermatocytes in initial phases of the first meiotic division, preleptotene, leptotene and zygotene. However, in pachytene phase they already showed a weak or negative staining. On the other hand, secondary spermatocytes, spermatids, spermatozoa and Sertoli cells were not stained. Based on this fact we suggest that ProT is present in the proliferative cycle in the final steps of G1 phase, throughout the S and G2 phases and in initial steps of the prophase.     

Progressive Increase In Polyamine Levels In 9l Cells in vitro During the Cell Cycle: Comparison Between Cells Isolated By Centrifugal Elutriation and Cells Grown In Synchrony

Centrifugal elutriation was used to separate 9L rat brain tumour cells into fractions enriched in the G₁, S, or G₂/M phases of the cell cycle. Cells enriched in early G₁, phase were recultured, grown in synchrony, and harvested periodically for analysis of their DNA distribution and polyamine content. Mathematical analysis of the DNA distributions indicated that excellent synchrony was obtained with low dissersion throughout the cell cycle. Polyamine accumulation began at the time of seeding, and intracellular levels of putrescine, spermidine, and spermine increased continuously during the cell cycle. In cells in the G₂/M phase of the cell cycle, putrescine and spermidine levels were twice as high as in cells in the G₁, phase. DNA distribution and polyamine levels were also analysed in cells taken directly from the various elutriation fractions enriched in G₁, S, or G₂/M. Because we did not obtain pure S or G₂/M populations by elutriation or by harvesting synchronized cells, a mathematical procedure—which assumed that the measured polyamine levels for any population were linearly related to the fraction of cells in the G₁, S, and G₂/M phases times the polyamine levels in these phases and that polyamine levels did not vary within these phases—was used to estimate ‘true’ phase-specific polyamine levels (levels to be expected if perfect synchrony were achieved). Estimated ‘true’ phase-specific polyamine levels calculated from the data obtained from cells either sorted by elutriation or obtained from synchronously growing cultures were very similar.     

Dynamics of cell cycle phase perturbations by trabectedin (ET-743) in nucleotide excision repair (NER)-deficient and NER-proficient cells, unravelled by a novel mathematical simulation approach

Abstract. Objectives: Trabectedin (ET‐743, Yondelis^®) is a natural marine product, with antitumour activity, currently in phase II/III clinical trials. Previous studies have shown that cells hypersensitive to ultraviolet (UV)‐rays because of nucleotide excision repair (NER) deficiency, were resistant to trabectedin. The purpose of this study was to investigate whether this resistance was associated with different drug‐induced cell cycle perturbations. Materials and Methods: An isogenic NER‐proficient cellular system (CHO‐AA8) and a NER‐deficient one (CHO‐UV‐96), lacking functional ERCC‐1, were studied. Flow cytometric assays showed progressive accumulation of cells in G₂ + M phase in NER‐proficient but not in NER‐deficient cells. Applying a computer simulation method, we realized that the dynamics of the cell cycle perturbations in all phases were complex. Results: Cells exposed to trabectedin during G₁ and G₂ + M first experienced a G₁ block, while those exposed in S phase were delayed in S and G₂ + M phases but eventually divided. In the presence of functional NER, exit from the G₁ block was faster; then, cells progressed slowly through S phase and were subsequently blocked in G₂ + M phase. This G₂ + M processing of trabectedin‐induced damage in NER‐proficient cells was unable to restore cell cycling, suggesting a difficulty in repairing the damage. Conclusions: This might be due either to important damage left unrepaired by previous G₁ repair, or that NER activity itself caused DNA damage, or both. We speculate that in UV‐96 cells repair mechanisms other than NER are activated both in G₁ and G₂ + M phases.     

Invading cancer cells are predominantly in G0/G1 resulting in chemoresistance demonstrated by real-time FUCCI imaging

Invasive cancer cells are a critical target in order to prevent metastasis. In the present report, we demonstrate real-time visualization of cell cycle kinetics of invading cancer cells in 3-dimensional (3D) Gelfoam® histoculture, which is in vivo-like. A fluorescence ubiquitination cell cycle indicator (FUCCI) whereby G₀/G₁ cells express a red fluorescent protein and S/G₂/M cells express a green fluorescent protein was used to determine the cell cycle position of invading and non-invading cells. With FUCCI 3D confocal imaging, we observed that cancer cells in G₀/G₁ phase in Gelfoam® histoculture migrated more rapidly and further than cancer cells in S/G₂/M phases. Cancer cells ceased migrating when they entered S/G₂/M phases and restarted migrating after cell division when the cells re-entered G₀/G₁. Migrating cancer cells also were resistant to cytotoxic chemotherapy, since they were preponderantly in G₀/G₁, where cytotoxic chemotherapy is not effective. The results of the present report suggest that novel therapy targeting G₀/G₁ cancer cells should be developed to prevent metastasis.     

Author index for volume 49

Using mouse thymocytes, mitogen-induced [³H]thymidine incorporation was compared with a recently developed flow-cytometric technique, based on acridine orange staining of cells, which differentiates the G₀ and G₁ phase of thymocytes. PHA induces a transient but considerable G₀-G₁ shift without any substantial proliferation. On the other hand, crude supernatants derived from Con A-stimulated human peripheral blood mononuclear cells induce only a minor G₀-G₁ shift and no proliferation. However, PHA in the presence of this supernatant induced an increased [³H]thymidine uptake in thymocytes and a shift from G₁ to S. These results support the current hypothesis that a factor present in Con A-activated supernatants in conjunction with PHA stimulation indeed facilitates the entrance of G₁ cells into the S phase. The flow-cytometric technique might be used in the study of the interaction of endogenous mediators with exogenous mitogenic agents in activating lymphocytes to proceed through the initial G₀-G₁ phases of the cell cycle.     

Noninvasive estimation of cell cycle phase and proliferation rate of human mesenchymal stem cells by phase-shifting laser microscopy

The simultaneous determination of the cell cycle phase of individual adherent mesenchymal stem cells (MSCs) using a fluorescence microscope after staining with 4′,6-diamidine-2′-phenylindole dihydrochloride and bromodeoxyuridine and the laser phase shift by phase-shifting laser microscopy (PLM) revealed that the laser phase shift of cells in the G₂/M phase was markedly higher than that of cells in the G₀/G₁ phase. Even in the synchronous cultures to G₀/G₁ and G₂/M cell cycle phases, the laser phase shift of the cells in the G₂/M phase was markedly higher than that of the cells in the G₀/G₁ phase. The analysis of the cultures of MSCs from different donors with the addition of FGF2 at different concentrations revealed that there was a marked negative correlation between the average phase shift and mean generation time. In conclusion, it is possible to estimate noninvasively the proliferation activity of MSCs population by measuring the phase shift using PLM.     

Microspectrophotometric measurements of DNA by automated computerized scanning in cycling cells of theChrysanthemum segetum shoot apex

Summary A new technique of exploitation of the data was proposed after DNA scanning microdensitometry. By using all of the measurements obtained from the seriated sections of a single nucleus, this method made it possible to estimate six characteristic parameters during the different phases of the cell cycle in the various shoot apical cells. The cells whose rate of proliferation was the highest showed the biggest variations of their nuclear and nucleolar volumes during the cell cycle. In the axial zone, where the cells have a slow cell cycle and display the longest duration of the G₁ phase, the volume occupied by dispersed DNA was greater than in the cells of the lateral zone and of the rib meristem, where the cell cycle and the G₁ phase were short. No matter what the cell type, the proportion of the dispersed and condensed DNA varied little when the G₁ and G₂ phases were compared. In the Z phase, characterized by a decondensation of the DNA, the mean DNA amount was 3.4 C. The evolution of the nuclear density during the interphase was also estimated. It is demonstrated that the main feature of the shoot apex zonation was the decondensation of the condensed DNA in the axial zone in both the G₁ and G₂ phases.     

Estrogen receptor alpha is cell cycle-regulated and regulates the cell cycle in a ligand-dependent fashion

Estrogen receptor alpha (ERα) has been implicated in several cell cycle regulatory events and is an important predictive marker of disease outcome in breast cancer patients. Here, we aimed to elucidate the mechanism through which ERα influences proliferation in breast cancer cells. Our results show that ERα protein is cell cycle-regulated in human breast cancer cells and that the presence of 17-β-estradiol (E2) in the culture medium shortened the cell cycle significantly (by 4.5 hours, P < 0.05) compared with unliganded conditions. The alterations in cell cycle duration were observed in the S and G₂/M phases, whereas the G₁ phase was indistinguishable under liganded and unliganded conditions. In addition, ERα knockdown in MCF-7 cells accelerated mitotic exit, whereas transfection of ERα-negative MDA-MB-231 cells with exogenous ERα significantly shortened the S and G₂/M phases (by 9.1 hours, P < 0.05) compared with parental cells. Finally, treatment of MCF-7 cells with antiestrogens revealed that tamoxifen yields a slower cell cycle progression through the S and G₂/M phases than fulvestrant does, presumably because of the destabilizing effect of fulvestrant on ERα protein. Together, these results show that ERα modulates breast cancer cell proliferation by regulating events during the S and G₂/M phases of the cell cycle in a ligand-dependent fashion. These results provide the rationale for an effective treatment strategy that includes a cell cycle inhibitor in combination with a drug that lowers estrogen levels, such as an aromatase inhibitor, and an antiestrogen that does not result in the degradation of ERα, such as tamoxifen.     

Differential stimulation of hepatitis C virus RNA translation by microRNA-122 in different cell cycle phases

Hepatitis C virus (HCV) replicates preferentially in the liver, and in most cases, the HCV infection becomes chronic and often results in hepatocellular carcinoma. When the HCV plus-strand RNA genome has been delivered to the cytosol of the infected cell, its translation is directed by the internal ribosome entry site (IRES) in the 5′-untranslated region (5′-UTR) of the viral RNA. Thereby, IRES activity is modulated by several host factors. In particular, the liver-specific microRNA-122 (miR-122) interacts with two target sites in the HCV 5′-UTR and stimulates HCV translation, thereby most likely contributing to HCV liver tropism. Here, we show that HCV IRES-dependent translation efficiency in the hepatoma cell line Huh7 is highest during the G₀ and G₁ phases of the cell cycle but significantly drops during S phase and even more in the G₂/M phase. The superimposed stimulation of HCV translation by ectopic miR-122 works best during G₀, G₁ and G₂/M phases but is lower during S phase. However, the levels of Ago2 protein do not substantially change during cell cycle phases, indicating that other cellular factors involved in HCV translation stimulation by miR-122 may be differentially expressed in different cell cycle phases. Moreover, the levels of endogenously expressed miR-122 in Huh7 cells are lowest in S phase, indicating that the predominant G₀/G₁ state of non-dividing hepatocytes in the liver facilitates high expression of the HCV genome and stimulation by miR-122, with yet-unknown factors involved in the differential extent of stimulation by miR-122.Key words: HCV, translation, miR-122, microRNA, miRNA, Ago, Ago2     

Cell-cycle dependency of radiosensitivity and mutagenesis in fertilized egg cells of rice,Oryza sativa L.

Summary In order to examine changes in survival and mutation rates during a cell cycle in higher plant, fertilized egg cells of rice were irradiated with X-rays at 2 h intervals for the first 36 h after pollination, i.e., at different phases of the first and second cell cycles. The most sensitive phase in lethality was late G₁ to early S, followed by late G₂ to M, which were more sensitive than the other phases. In both M₁ and M₂ generations, sterile plants appeared most frequently when fertilized egg cells were irradiated at G₂ and M phases. Different kinds of mutated characters gave rise to the respective maximum mutation rates at different phases of a cell cycle: namely, albino and viridis were efficiently induced at early G₁, xantha at early S, short-culm mutant at mid G₂, heading-date mutant at M to early G₁. The present study suggests the possibility that the differential mutation spectrums concerning agronomic traits are obtained by selecting the time of irradiation after pollination.     

Conservative homologous recombination preferentially repairs DNA double-strand breaks in the S phase of the cell cycle in human cells

DNA double-strand breaks (DSBs) are repaired by either homologous recombination (HR) or non-homologous end joining (NHEJ) in mammalian cells. Repair with NHEJ or HR using single-strand annealing (SSA) often results in deletions and is generally referred to as non-conservative recombination. Error-free, conservative HR involves strand invasion and requires a homologous DNA template, and therefore it is generally believed that this type of repair occurs preferentially in the late S, G₂ and M phases of the cell cycle, when the sister chromatid is available. There are several observations supporting this hypothesis, although it has not been tested directly. Here, we synchronize human SW480SN.3 cells in the G₁/G₀ (with serum starvation), S (with thymidine block) and M (with nocodazole) phases of the cell cycle and investigate the efficiency of conservative HR repair of an I-SceI-induced DSB. The frequency of HR repair of DSBs was 39 times higher in S-phase cells than in M-phase cells and 24-fold higher than in G₁/G₀ cells. This low level of conservative HR occurs even though a homologous template is present within the recombination substrate. We propose that this can be explained by an absence of recombination proteins outside the S phase or alternatively that there maybe factors that suppress HR in G₁/G₀ and M. Furthermore, we found that HR repair of DSBs involves short tract gene conversion in all the phases of the cell cycle. This indicates that the same pathway for conservative HR is employed in the repair of DSBs regardless of phase of the cell cycle and that only the frequency is affected.     

Impact of cell cycle dynamics on pathology recognition: Raman imaging study

Confocal Raman imaging combined with fluorescence‐activated cell sorting was used for in vitro studies of cell cultures to look at biochemical differences between the cells in different cell phases. To answer the question what is the impact of the cell cycle phase on discrimination of pathological cells, the combination of several factors was checked: a confluency of cell culture, the cell cycle dynamics and development of pathology. Confluency of 70% and 100% results in significant phenotypic cell changes that can be also diverse for different batches. In 100% confluency cultures, cells from various phases become phenotypically very similar and their recognition based on Raman spectra is not possible. For lower confluency, spectroscopic differences can be found between cell cycle phases (G₀/G₁, S and G₂/M) for control cells and cells incubated with tumor necrosis factor alpha (TNF‐α), but when the mycotoxin cytochalasin B is used the Raman signatures of cell phases are not separable. Generally, this work shows that heterogeneity between control and inflamed cells can be bigger than heterogeneity between cell cycle phases, but it is related to several factors, and not always can be treated as a rule.      

Quantitative Analysis of Centromeric FISH Spots during the Cell Cycle by Image Cytometry

Two-color fluorescence in situ hybridization (FISH) with chromosome enumeration DNA probes specific to chromosomes 7, 11, 17, and 18 was applied to CAL-51 breast cancer cells to examine whether the fluorescence intensity of FISH spots was associated with cell cycle progression. The fluorescence intensity of each FISH spot was quantitatively analyzed based on the cell cycle stage determined by image cytometry at the single-cell level. The spot intensity of cells in the G₂ phase was larger than that in the G_0/1 phase. This increased intensity was not seen during the early and mid S phases, whereas the cells in the late S phase showed significant increases in spot intensity, reaching the same level as that observed in the G₂ phase, indicating that alpha satellite DNA in the centromeric region was replicated in the late S phase. Thus, image cytometry can successfully detect small differences in the fluorescence intensities of centromeric spots of homologous chromosomes. This combinational image analysis of FISH spots and the cell cycle with cell image cytometry provides insights into new aspects of the cell cycle. This is the first report demonstrating that image cytometry can be used to analyze the fluorescence intensity of FISH signals during the cell cycle.     

Hdm2- and proteasome-dependent turnover limits p21 accumulation during S phase

Double-strand DNA breaks detected in different phases of the cell cycle induce molecularly distinct checkpoints downstream of the ATM kinase. p53 is known to induce arrest of cells in G₁ and occasionally G₂ phase but not S phase following ionizing radiation, a time at which the MRN complex and cdc25-dependent mechanisms induce arrest. Our understanding of how cell cycle phase modulates pathway choice and the reasons certain pathways might be favored at different times is limited. In this report, we examined how cell cycle phase affects the activation of the p53 checkpoint and its ability to induce accumulation of the cdk2 inhibitor p21. Using flow cytometric tools and centrifugal elutriation, we found that the p53 response to ionizing radiation is largely intact in all phases of the cell cycle; however, the accumulation of p21 protein is limited to the G₁ and G₂ phase of the cell cycle because of the activity of a proteasome-dependent p21 turnover pathway in S-phase cells. We found that the turnover of p21 was independent of the SCF^skp2 E3 ligase but could be inhibited, at least in part, by reducing hdm2, although this depended on the cell type studied. Our results suggest that there are several redundant pathways active in S-phase cells that can prevent the accumulation of p21.     

Conjugation Between G1 and G2 Phase Cells in Paramecium tetraurelia1

Cells of Paramecium tetraurelia, stock hr^d, cultured in a micro-capillary containing 1 μl fresh culture medium, expressed mating activity through the whole cell cycle. Mating-reactive G₂ phase cells can conjugate with cells of other phases. The G₂ phase cells, which have double (4C) the normal micronuclear DNA content, undergo pre-meiotic DNA synthesis when conjugated with G₁ phase cells. The micronucleus of the progeny from the cross between a G₁ and a G₂ cell becomes triploid.     

Developing a cyclin blueprint as a tool for mapping the cell cycle in GS-NS0

The cell cycle is at the center of growth, productivity, and death of mammalian cell cultures. There exists a need to identify and quantify major landmarks in the cell cycle of industrially relevant mammalian cell lines and its association with productivity; central for designing productivity optimization strategies. Herein, we studied the expression of three cyclins, under both perturbed and unperturbed growth, by flow cytometry in batch cultures of GS-NS0. The perturbed systems involved two different DNA synthesis inhibitors, thymidine and dimethyl sulfoxide (DMSO). This approach enables the establishment of characteristic cyclin profiles, timings, and thresholds. In particular, two G₁ class cyclins (D1 and E1), and one G₂ cyclin (B1) were investigated. Cyclin B1 showed a clear cell cycle phase-specific expression increasing during G₂ phase where it was approximately 40% higher when compared to G₁ phase. Similarly, cyclin E1 showed a clear pattern being expressed approximately 10% higher in G₁ compared to G₂ phase and decreased through S phase. Cyclin D1 expression was fairly invariable throughout the cell cycle phases. The observed patterns provide a blueprint of the cell line's cell cycle, which can be used for the development of biologically accurate and experimentally validated distributed cell cycle models.     

Flow cytometric BrdUrd-pulse-chase study of X-ray-induced alterations in cell cycle progression

To better understand how the flow cytometric bromodeoxyuridine (BrdUrd)-pulse-chase method detects perturbed cell kinetics we applied it to measure cell cycle progression delays following exposure to ionizing radiation. Since this method will allow both the use of asynchronous cell populations and the determination of the alterations in cell cycle progression specific to cells irradiated in given cell cycle phases, it has a significant advantage over laborious synchronization methods. Exponentially growing Chinese hamster ovary (CHO) K1 cells were irradiated with graded doses of X-rays and pulse-labelled with BrdUrd immediately thereafter. Cells were subcultured in a BrdUrd-free medium for various time intervals and prepared for flow cytometric analysis. Of five flow cytometric parameters examined, only those that involved cell transit through G₂, i.e. the fraction of BrdUrd-negative G₂ cells and the fraction of BrdUrd-positive cells that had not divided, showed radiation dose-dependent delays. The magnitude of the effects indicates that the cells irradiated in G₂ and in S are equally delayed. S phase transit of cells irradiated in S or in G₁ did not appear to be affected. There were apparent changes in flow of cells out of G₁, which could be explained by the delayed entry of G₂ cells into the compartment because of G₂ arrest. Thus, in asynchronous cells the method was able to detect G₂ delay in those cells irradiated in S and G₂ phases and demonstrate the absence of cell-cycle delays in other phases.     

Expression of the rat prothymosin alpha gene during T-lymphocyte proliferation and liver regeneration.

Prothymosin alpha (ProT alpha) is a widely distributed acidic protein whose function has been related to cell proliferation. We have analyzed the expression of the rat ProT alpha gene in several proliferative systems: concanavalin A (ConA)/interleukin-2-stimulated thymocytes, ConA-stimulated splenic T-lymphocytes, and hepatocytes proliferating during liver regeneration. In these systems, ProT alpha mRNA was detected in all stages of the cell cycle, with maximal increments (2-4-fold) at the beginning of the S phase. By contrast, the mRNAs for proliferating cell nuclear antigen/cyclin and histone H3, two cell-cycle-regulated proteins, were hardly detected in resting cells but increased notably at the G1/S boundary and in the S phase, respectively. Treatment of T-cells with the calcium ionophore A23187 increased ProT alpha mRNA levels 2.5-fold, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, had no effect on ProT alpha gene expression. Incubation of ConA-stimulated T-cells with hydroxyurea, a DNA synthesis inhibitor, did not decrease the levels of ProT alpha mRNA, indicating that its expression is independent of DNA synthesis. These findings suggest that ProT alpha is required throughout all the stages of the cell cycle, resembling a constitutively expressed gene rather than one strictly involved in cell proliferation.     

Cell cycle: Regulatory events in G1 → S transition of mammalian cells

A cell divides into two daughter cells by progressing serially through the precisely controlled G₁, S, G₂, and M phases of the cell cycle. The crossing of the G₁/S border, which is marked by the initiation of DNA synthesis, represents commitment to division into two complete cells. Beyond this critical point no further external signals are required. We now have more comprehensive knowledge of the temporal sequence of systems at this key transition from G₁ to S—growth factor responses, a cascade of kinase reactions, activation of cyclins and their associated kinases, and oncogene and tumor suppressor gene products. Furthermore, we know that the absolute requirement for calcium and the timing of events associated with calmodulin and the 68 kDa calmodulin-binding protein are consistent with overall Ca⁺⁺/calmodulin control of all steps from the response to growth factors in G₁ to DNA replication in S phase. We now have to sort out the inter-relationships of myriad control proteins and their relation to the Ca⁺⁺/calmodulin-dependent controls—Which are causes? Which are effects? And which are parallel processes? The answers will be important, as they represent both a much deeper understanding of this key process of life and an important opportunity for improving therapeutic medicine.