The Effect of Dia2 Protein Deficiency on the Cell Cycle,Cell Size,and Recruitment of Ctf4 Protein in Saccharomyces cerevisiae

Cells have evolved elaborate mechanisms to regulate DNA replication machinery and cell cycles in response to DNA damage and replication stress in order to prevent genomic instability and cancer. The E3 ubiquitin ligase SCF^Dia2 in S. cerevisiae is involved in the DNA replication and DNA damage stress response, but its effect on cell growth is still unclear. Here, we demonstrate that the absence of Dia2 prolongs the cell cycle by extending both S- and G2/M-phases while, at the same time, activating the S-phase checkpoint. In these conditions, Ctf4—an essential DNA replication protein and substrate of Dia2—prolongs its binding to the chromatin during the extended S- and G2/M-phases. Notably, the prolonged cell cycle when Dia2 is absent is accompanied by a marked increase in cell size. We found that while both DNA replication inhibition and an absence of Dia2 exerts effects on cell cycle duration and cell size, Dia2 deficiency leads to a much more profound increase in cell size and a substantially lesser effect on cell cycle duration compared to DNA replication inhibition. Our results suggest that the increased cell size in dia2∆ involves a complex mechanism in which the prolonged cell cycle is one of the driving forces.     

THE EPIDERMAL CELL KINETIC RESPONSE TO ULTRAVIOLET B IRRADIATION COMBINES REGENERATIVE PROLIFERATION AND CARCINOGEN ASSOCIATED CELL CYCLE DELAY

The cell cycle traverse of epidermal basal cells 24 h after in vivo exposure of ultraviolet B (UVB) irradiation was studied by immunochemical staining of incorporated bromodeoxyuridine (BrdU) and bivariate BrdU/DNA flow cytometric analysis. The results were compared with the cell kinetic patterns following topical application of the skin carcinogen methylnitrosourea (MNU) as well as the skin irritant cantharidin. Hairless mice were injected intraperitoneally with BrdU 24 h after treatment of their back skin with either a minimal erythema dose of UVB, or a single application of MNU or cantharidin dissolved in acetone. The cell cycle traverse of the BrdU-labelled cohorts of epidermal basal cells were then followed for the subsequent 12 h. At 6 h after BrdU-injection, when all labelled cells in the control group as well as in the cantharidin group had left the S phase, the bivariate distributions of the UVB-exposed and the MNU group showed that BrdU-positive cells were still present in S phase. Hence, UVB irradiation, similar to the carcinogen MNU, prolonged the S phase duration in some of the basal cells. At 12 h after pulse labelling, however, BrdU-positive cells from UVB-exposed mice were re-entering S phase from G1 phase, indicating that UVB irradiation induced a shortening of the cell cycle time as well, similar to the response observed after cantharidin. The present data can not tell whether these cells also were delayed in S phase. Thus, the cell cycle traverse in hairless mouse epidermis 24 h after in vivo exposure to UVB seemed to be a combination of the cell kinetic effects following chemical skin carcinogens and skin irritants. UVB irradiation induced both a delay in transit time through S phase, probably due to DNA damage and subsequent repair, as well as a reduction in the total cell cycle time consistent with rapid regenerative proliferation.     

Synthesis and identification of small molecules that potently induce apoptosis in melanoma cells through G1 cell cycle arrest

Late-stage malignant melanoma is a cancer that is refractory to current chemotherapeutic treatments. The average survival time for patients with such a diagnosis is 6 months. In general, the vast majority of anticancer drugs operate through induction of cell cycle arrest and cell death in either the DNA synthesis (S) or mitosis (M) phase of the cell cycle. Unfortunately, the same mechanisms that melanocytes possess to protect cells from DNA damage often confer resistance to drugs that derive their toxicity from S or M phase arrest. Described herein is the synthesis of a combinatorial library of potential proapoptotic agents and the subsequent identification of a class of small molecules (triphenylmethylamides, TPMAs) that arrest the growth of melanoma cells in the G1 phase of the cell cycle. Several of these TPMAs are quite potent inducers of apoptotic death in melanoma cell lines (IC(50) approximately 0.5 muM), and importantly, some TPMAs are comparatively nontoxic to normal cells isolated from the bone marrow of healthy donors. Furthermore, the TPMAs were found to dramatically reduce the level of active nuclear factor kappa-B (NFkappaB) in the cell; NFkappaB is known to be constitutively active in melanoma, and this activity is critical for the proliferation of melanoma cells and their evasion of apoptosis. Compounds that reduce the level of NFkappaB and arrest cells in the G1 phase of the cell cycle can provide insights into the biology of melanoma and may be effective antimelanoma agents.     

High-throughput cell cycle synchronization using inertial forces in spiral microchannels

Efficient synchronization and selection of cells at different stages of the cell replication cycle facilitates both fundamental research and development of cell cycle-targeted therapies. Current chemical-based synchronization methods are unfavorable as these can disrupt cell physiology and metabolism. Microfluidic systems developed for physical cell separation offer a potential alternative over conventional cell synchronization approaches. Here we introduce a spiral microfluidic device for cell cycle synchronization, using the combined effects of inertial forces and Dean drag force. By exploiting the relationship between cell diameter and cell cycle (DNA content/ploidy), we have successfully fractionated several asynchronous mammalian cell lines, as well as primary cells comprising bone marrow-derived human mesenchymal stem cells (hMSCs), into enriched subpopulations of G0/G1 (>85%), S, and G2/M phases. This level of cell cycle enrichment is comparable to existing microfluidic systems, but the throughput (～ 15 × 10(6) cells per h) and viability (～ 95%) of cells thus synchronized are significantly greater. Further, this platform provides rapid collection of synchronized cells or of diameter-sorted cells post-separation, to enable diverse applications in the study and manipulation of cell proliferation.     

Anti-proliferation effects of ethanolic extracts from Sophora moorcroftiana seeds on human hepatocarcinoma HepG2 cell line

Previous studies have shown that the ethanolic extracts from Sophora moorcroftiana seeds (ee-Sms) have in vitro anticancer properties. The anti-proliferation effects of ee-Sms on HepG2 cells were assessed by MTT assay and cell cycle analysis. Total cell proteins were separated by two-dimensional electrophoresis (2-DE), and protein spots with more than two-fold difference were analysed by MALDI-TOF/TOF-MS. MTT assay showed that the anti-proliferation of ee-Sms demonstrates dose- and time dependently. HepG2 cells were treated with ee-Sms at 1.30 mg/mL for 48 h induced cell cycle arrest in S phase. The differentially-expressed proteins were involved in DNA repair, cell proliferation, cell metabolism and immunoreaction. This study sheds new insights into the molecular mechanisms underlying the anti-proliferation properties of ee-Sms in HepG2 cells.     

Induction of persistent double strand breaks following multiphoton irradiation of cycling and G1-arrested mammalian cells-replication-induced double strand breaks

DNA double strand breaks (DSBs) are amongst the most deleterious lesions induced within the cell following exposure to ionizing radiation. Mammalian cells repair these breaks predominantly via the nonhomologous end joining pathway which is active throughout the cell cycle and is error prone. The alternative pathway for repair of DSBs is homologous recombination (HR) which is error free and active during S- and G2/M-phases of the cell cycle. We have utilized near-infrared laser radiation to induce DNA damage in individual mammalian cells through multiphoton excitation processes to investigate the dynamics of single cell DNA damage processing. We have used immunofluorescent imaging of gamma-H2AX (a marker for DSBs) in mammalian cells and investigated the colocalization of this protein with ATM, p53 binding protein 1 and RAD51, an integral protein of the HR DNA repair pathway. We have observed persistent DSBs at later times postlaser irradiation which are indicative of DSBs arising at replication, presumably from UV photoproducts or clustered damage containing single strand breaks. Cell cycle studies have shown that in G1 cells, a significant fraction of multiphoton laser-induced prompt DSBs persists for > 4 h in addition to those induced at replication.     

Protein profiles of the Chinese hamster ovary cells in the resting and proliferating stages.

Identification and characterization of the proteins that regulate the transition from the resting stage (G0) through G1 to S phase of the cell cycle are of central importance to understand the control of cell proliferation and chromosome replication. Unlike in lower organisms, where relatively small numbers of key factors are involved in this process, the factors involved in the same control mechanisms in mammalian systems are much more complex. Furthermore, accumulating lines of evidence now suggest that the nuclear matrix and chromatin organization also play an essential role for the cell cycle control in mammalian cells. To gain a better understanding of the overall dynamics and changes of the protein factors in the context of matrix/chromatin organization, we examined the protein profiles of the Chinese hamster ovary (CHO) cells in different cell cycle compartments. The methods used in this study included subcellular fractionations (cytosol, nuclear extraction, chromatin, and nuclear matrix), two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), silver staining, and immunoblotting. As expected, significant changes of protein profiles were observed when cells entered into proliferating stages from G0. Among approximately 1200 protein spots analyzed by 2-D PAGE, at least 12 showed marked increase or decrease at this transitional period. Further cell-cycle progression from G1 to S phase showed less dramatic changes of overall protein protile. However, the profile of certain proteins showed rather dramatic changes of their subcellular localization during this transitional period. In particular, the levels of proliferating cell nuclear antigen (PCNA) in the nuclear matrix and chromatin dramatically increased in mid-G1 and in the beginning of S phase, respectively, while the overall PCNA level was relatively constant throughout the cell cycle.     

Immune diversity and genomic stability: opposite goals but similar paths.

DNA damage response mechanisms serve to protect cells from exogenous and endogenous DNA damaging agents with the aim of maintaining genomic stability. In contrast, the generation of an efficient immune response requires the creation of a repertoire of distinct immunoglobulin and T cell receptor genes able to recognise the huge array of antigens that may be encountered in a lifetime. Surprisingly, cells have exploited the same mechanisms used to maintain genomic integrity to create genetic diversity during immune development. Here, we review the damage response mechanisms operating on DNA double strand breaks and their function during development of the immune response. We discuss disorders that are associated with immunodeficiency and defective responses to the presence of DNA double strand breaks.     

Cell cycle protein profile of the hepatic stellate cells(HSCs)in dimethylnitrosamine-induced rat hepatic fibrosis

Cell cycle regulating proteins are known to have close relation with the proliferation of the mammalian cells. In injured liver, the number of HSCs is increased from proliferation. However, the expression of cell cycle proteins of HSCs during proliferation remains unevaluated. Therefore, cell cycle protein profiles of HSCs were studied in dimethyl-nitrosamine (DMN)-induced rat liver fibrosis model. Sprague-Dawley rats were intraperitoneally injected of DMN and the animals were sacrificed every week up to 4 weeks. HSCs were separated and the number of the cells in S phase was counted to evaluate the cell proliferation by flow cytometry. The expression of cyclin A, cyclin B, cyclin D1, cdk2, cdk4, cdc2, proliferating cell nuclear antigen (PCNA), p21(Cip/WAF1), and p27 was examined with immunoblotting analysis. Portion of S-phase cells peaked 7days after DMN injection. At that time, cyclin A, and PCNA showed significant increase in HSCs compared to untreated HSCs (114% and 116%, respectively, P<0.001). p21(Cip/WAF1) was decreased significantly in DMN-treated HSCs compared to control cells (88%, P<0.001). The increase of cyclin A, and PCNA and the decrease of p21(Cip/WAF1) seem to play important roles in the proliferation of HSCs during the early period of DMN treatment.     

Embryonic stem cell markers

Embryonic stem cell (ESC) markers are molecules specifically expressed in ES cells. Understanding of the functions of these markers is critical for characterization and elucidation for the mechanism of ESC pluripotent maintenance and self-renewal, therefore helping to accelerate the clinical application of ES cells. Unfortunately, different cell types can share single or sometimes multiple markers; thus the main obstacle in the clinical application of ESC is to purify ES cells from other types of cells, especially tumor cells. Currently, the marker-based flow cytometry (FCM) technique and magnetic cell sorting (MACS) are the most effective cell isolating methods, and a detailed maker list will help to initially identify, as well as isolate ESCs using these methods. In the current review, we discuss a wide range of cell surface and generic molecular markers that are indicative of the undifferentiated ESCs. Other types of molecules, such as lectins and peptides, which bind to ESC via affinity and specificity, are also summarized. In addition, we review several markers that overlap with tumor stem cells (TSCs), which suggest that uncertainty still exists regarding the benefits of using these markers alone or in various combinations when identifying and isolating cells.     

The mechanism of cytotoxicity of methylmercury: Inhibition of progression through the S phase of the cell cycle

The effect of methylmercury (MeHg) on progression of the murine erythroleukemic cell (MELC) through the cell cycle was analyzed by flow cytometry (FCM). Exposure in vitro to 5.0-10.0 μmol dm^?3 MeHg for 6h resulted in a dose-dependent decrease in the rate of cell replication, apparently as a result of inhibition of DNA synthesis (rate of passage through the S phase of the cell cycle). Thus, only a modest accumulation of cells with a G₂/M (4n) DNA content was observed. At or above 10 μmol dm^?3 MeHg, progression through all phases of the cell cycle was blocked. FCM revealed a dose-dependent increase in cellular refractive index (90° light scatter), decrease in apparent cell volume (axial light loss), and increase in resistance to non-ionic detergent (NP-40)-mediated cytolysis indicative of fixation (protein denaturation, cross-linking, etc.) of the plasma membrane/cytoplasm complex. The data indicate DNA synthesis as the primary target of MeHg cytotoxicity.     

Growth Characteristics of Human Adipose-Derived Stem Cells During Long Time Culture Regulated by Cyclin A and Cyclin D1

Abundant and less passaged cells are highly expected in clinical application since repeated subculture reduces stem cell characteristics. Long time culture of stem cells without passage is therefore needed. The growth and cell viability of human adipose-derived stem cells (hADSCs) were investigated by live/dead staining, cck-8 kits, and hemocytometer every day in 30?days of culture. The stem cell characteristics of hADSCs at the beginning and the end of culture were detected by flow cytometry and histochemical staining. hADSCs can be cultured up to the 30th day in one passage while maintaining high level cell viability and their stem cell characteristics. In addition, the cells displayed two plateau phases and three logarithmic phases during 1?month of culture. Increasing expression of cyclin A at protein level resulted in an increase in the percentage of hADSCs in the S and G₂/M phases, while decreasing protein level of cyclin D1 induced a decline in the proportion of hADSCs in the G₀/G₁ phase, regulating cells to move into rapid proliferation. This study demonstrates that a great quantity of hADSCs can be obtained in vitro by prolonging the culture time of each passage. And cyclin A and cyclin D1 affect the distribution of cell cycle and regulate the growth of hADSCs.     

ATR Kinase Activity Limits Mutagenesis and Promotes the Clonogenic Survival of Quiescent Human Keratinocytes Exposed to UVB Radiation

The ATR protein kinase has well-described roles in maintaining genomic integrity during the DNA synthesis phase of the cell cycle. However, ATR function in cells that are not actively replicating DNA remains largely unexplored. Using HaCaT and telomerase-immortalized human keratinocytes maintained in a confluent, nonreplicating state in vitro, ATR was found to be robustly activated in response to UVB radiation in a manner dependent on the nucleotide excision repair factor and DNA translocase XPB. Inhibition of ATR kinase activity under these conditions negatively impacted acute cell survival and cytotoxicity and severely inhibited the ability of UVB-irradiated HaCaT keratinocytes to proliferate upon stimulation with growth factors. Furthermore, ATR kinase inhibition in quiescent HaCaT keratinocytes potentiated UVB mutagenesis at the hypoxanthine phosphoribosyltransferase locus. Though ATR inhibition did not impact the rate of removal of cyclobutane pyrimidine dimers from genomic DNA, elevated levels of PCNA mono-ubiquitination and chromatin-associated PCNA and RPA indicate that excision gap-filling synthesis was altered in the absence of ATR signaling. These results indicate that the ATR kinase plays important roles in preventing mutagenesis and in promoting the proliferative potential of quiescent keratinocytes exposed to UVB radiation.