Effects of guanine nucleotide depletion on cell cycle progression in human T lymphocytes

Depletion of guanine nucleotide pools after inhibition of inosine monophosphate dehydrogenase (IMPDH) potently inhibits DNA synthesis by arresting cells in G1 and has been shown to induce the differentiation of cultured myeloid and erythroid cell lines, as well as chronic granulocytic leukemic cells after blast transformation. Inhibitors of IMPDH are also highly effective as immunosuppressive agents. The mechanism underlying these pleiotropic effects of depletion of guanine nucleotides is unknown. We have examined the effects of mycophenolic acid (MPA), a potent IMPDH inhibitor, on the cell cycle progression of activated normal human T lymphocytes. MPA treatment resulted in the inhibition of pRb phosphorylation and cell entry into S phase. The expression of cyclin D3, a major component of the cyclin-dependent kinase (CDK) activity required for pRb phosphorylation, was completely abrogated by MPA treatment of T cells activated by interleukin-2 (IL-2) and leucoagglutinin (PHA-L), whereas the expression of cyclin D2, CDK6, and CDK4 was more mildly attenuated. The direct kinase activity of a complex immunoprecipitated with anti-CDK6 antibody was also inhibited. In addition, MPA prevented the IL-2-induced elimination of p27(Kip1), a CDK inhibitor, and resulted in the retention of high levels of p27(Kip1) in IL-2/PHA-L-treated T cells bound to CDK2. These results indicate that inhibition of the de novo synthesis of guanine nucleotides blocks the transition of normal peripheral blood T lymphocytes from G0 to S phase in early- to mid-G1 and that this cell cycle arrest results from inhibition of the induction of cyclin D/CDK6 kinase and the elimination of p27(Kip1) inhibitory activity.     

ALG gene expression and cell cycle progression

The evolutionarily conserved ALG genes function in the dolichol pathway in the synthesis of the lipid-linked oligosaccharide precursor for protein N-glycosylation. Increasing evidence suggests a role for these genes in the cell cycle. In Saccharomyces cerevisiae, coordinate regulation of the ALG genes makes up the primary genomic response to growth stimulation; several features of the ALG genes' expression resemble mammalian early growth response genes. However, only the first gene in the pathway, ALG7, is downregulated in response to an antimitogenic signal that leads to cell cycle arrest and differentiation, suggesting that selective inhibition of the first gene may be sufficient to regulate the dolichol pathway for the withdrawal from the cell cycle. The availability of mutants in the early essential ALG genes has established functional relationships between these genes' expression and G1/S transition, budding, progression through G2 and withdrawal from the cell cycle. Analysis of the regulation of ALG7 has provided insights into how this gene's expression is controlled at the molecular level. Recent studies have also begun to reveal how ALG7 expression is linked to cell cycle arrest in response to antimitogenic cues and have identified G1 cyclins as some of its downstream targets. Since the functions of the ALG genes appear to be as conserved among eukaryotes as the cell cycle machinery, it is likely that these genes play a similar role in mammalian cell proliferation and differentiation.     

Effect of beta-carotene on cell cycle progression of human fibroblasts

The uptake of beta-carotene (BC) and its effect on the cell cycle progression of normal human fibroblasts in primary culture were investigated by using two different delivery methods: exposure to BC solubilized in the organic solvent tetrahydrofuran (THF) or to BC incorporated into dipalmitoylphosphatidylcholine (DPPC) liposomes. Cell cycle progression was evaluated by immunofluorescence detection and flow cytometric analysis of the proliferating cell nuclear antigen (PCNA). In contrast to THF, which induced a marked reduction in the number of cells in S phase and in the extent of PCNA immunolabeling, DPPC liposomes proved to be an effective delivery system that does not interfere with cell proliferation. Cellular uptake of 0.23 nmol/10(6) cells was found after 24 h incubation in BC-containing DPPC liposomes. This value increased to 1.2 nmol/10(6) cells after 72 h. After the first day of incubation, the number of cells in S phase was reduced by approximately 50%, with a consequent accumulation of cells in G1 phase. This effect was maintained up to 3 days incubation, with no detectable effects on cell viability. This cell cycle delay was found to be reversible, returning the percentage of cells in S phase to the control value 24 h after removal of BC from the medium. In order to determine whether the activity of BC could be attributed to the molecule itself or to its conversion into retinoids, the production of BC metabolites was assessed. Analysis of cellular levels of retinoids failed to demonstrate the presence of retinal, retinol, retinoic acid or retinyl esters during an incubation period of 6 days. These results suggest that in normal human fibroblasts, BC induces a cell cycle delay in the G1 phase and that this effect is independent of conversion to known retinoids.     

Taurolidine联合X射线对小鼠恶性黑色素瘤细胞周期进程的影响

目的:观察taurolidine联合X射线照射对小鼠恶性黑色素瘤(B16-4A5和B16-F10)细胞周期进程的影响,探讨其诱导肿瘤细胞凋亡的发生机制.方法:选择B16-4A5和B16-F10细胞系按给药浓度随机分为4组,taurolidine剂量分别为0、25、50和100 μmol·L-1,同时进行1、2和4 Gy X射线照射,采用流式细胞术检测细胞凋亡率和细胞周期,Western blotting分析cyclin B、cdc2和caspase-3的表达.结果:与25 μmol·L-1 taurolidine组比较,50和100 μmol·L-1 taurolidine组诱导B16-4A5和B16-F10细胞发生G0/G1期阻滞,细胞数分别升高54.9%、73.7%和36.8%、55.5%(P＜0.05); 50 μmol·L-1 taurolidine联合2和4 Gy X射线照射组,细胞G2/M期阻滞消除,细胞数分别降低52.1%、44.2%和59.3%、52.7%(P＜0.05).与对照组、单纯taurolidine组及单纯照射组比较,联合4 Gy X射线照射组cyclin B和cdc2的表达降低,caspase-3的表达升高(P＜0.05).结论:taurolidine联合X射线照射可去除G2/M期阻滞,可选择地抑制肿瘤细胞cyclin B和cdc2的表达、增强caspase-3的表达,共同诱导细胞凋亡.     

Control of cell cycle progression by fibronectin matrix architecture

Developmental patterning and differentiation, maintenance of parenchymal cell function, and the size, shape, and invasiveness of tumors are all orchestrated by cell interactions with the extracellular matrix. Here we show that the fibrillar structure of fibronectin (FN) matrix encodes essential regulatory cues and controls cell proliferation and signaling through changes in matrix architecture. A matrix assembled from native FN stimulated cell growth. In contrast, a mutant FN (FNDeltaIII1-7) that contains all known cell binding motifs but forms a structurally distinct matrix inhibited progression from G0/G1 into S phase. Furthermore, FNDeltaIII1-7 suppressed the stimulatory capacity of native FN and induced different levels of tyrosine phosphorylation of pp125(FAK). The differential effects on cell growth were ablated by blocking formation of matrix fibrils. Thus, modification of matrix architecture provides a novel approach to control cell proliferation.     

A link between cyclin A expression and adhesion-dependent cell cycle progression

Cell adhesion has an essential role in regulating proliferation during the G1 phase of the cell cycle, and loss of this adhesion requirement is a classic feature of oncogenic transformation. The appearance of cyclin A messenger RNA and protein in late G1 was dependent on cell adhesion in both NRK and NIH 3T3 fibroblasts. In contrast, the expression of Cdc2, Cdk2, cyclin D1, and cyclin E was independent of adhesion in both cell lines. Transfection of NRK cells with a cyclin A complementary DNA resulted in adhesion-independent accumulation of cyclin A protein and cyclin A-associated kinase activity. These transfected cells also entered S phase and complete multiple rounds of cell division in the absence of cell adhesion. Thus, cyclin A is a target of the adhesion-dependent signals that control cell proliferation.     

Function of trehalose and glycogen in cell cycle progression and cell viability in Saccharomyces cerevisiae

Trehalose and glycogen accumulate in Saccharomyces cerevisiae when growth conditions deteriorate. It has been suggested that aside from functioning as storage factors and stress protectants, these carbohydrates may be required for cell cycle progression at low growth rates under carbon limitation. By using a mutant unable to synthesize trehalose and glycogen, we have investigated this requirement of trehalose and glycogen under carbon-limited conditions in continuous cultures. Trehalose and glycogen levels increased with decreasing growth rates in the wild-type strain, whereas no trehalose or glycogen was detected in the mutant. However, the mutant was still able to grow and divide at low growth rates with doubling times similar to those for the wild-type strain, indicating that trehalose and glycogen are not essential for cell cycle progression. Nevertheless, upon a slight increase of extracellular carbohydrates, the wild-type strain degraded its reserve carbohydrates and was able to enter a cell division cycle faster than the mutant. In addition, wild-type cells survived much longer than the mutant cells when extracellular carbon was exhausted. Thus, trehalose and glycogen have a dual role under these conditions, serving as storage factors during carbon starvation and providing quickly a higher carbon and ATP flux when conditions improve. Interestingly, the CO2 production rate and hence the ATP flux were higher in the mutant than in the wild-type strain at low growth rates. The possibility that the mutant strain requires this steady higher glycolytic flux at low growth rates for passage through Start is discussed.     

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.     

Fibronectin matrix regulates activation of RHO and CDC42 GTPases and cell cycle progression

This research concerned the use of mental rotation in recognizing rotated objects. Instead of the classic Shepard's paradigm in which subjects were still while observing rotated objects, here subjects had to move (or imagine moving) around stationary three-dimensional objects put in the middle of the trajectory. Thus, depending on the viewing positions, such objects were seen under six different perspectives (from 30 degrees to 180 degrees). The latter task has been thought to be closer to everyday life in which we obtain information regarding objects from their spatial properties. The results do not follow the classic rules of mental rotation of an object predicting a linear increase of the time needed to recognize distorted objects as a function of their angular displacement. They also differ from data in the literature about spatial imagery showing that access to spatial information is facilitated more when people actually move through a path than when they imagine moving. A probable explanation of this difference from the literature is discussed in relation to the particular involvement of the body in the experimental task.     

Development of new antitumor reagents which block the cell cycle progression]

Most tumor cells have abnormalities in the cell-cycle regulation system. Cyclin-dependent kinases (cdk) are major regulatory molecules for the cell cycle control. Therefore, cell cycle inhibitors, especially cdk-inhibitors, may be a promising new type of antitumor drug. In this review, low-molecular-weight cell-cycle inhibitors isolated from mainly microorganisms are described. As the activation of cdk is caused by the association with cyclins as well as by their phosphorylation and dephosphorylation, kinase inhibitors and phosphatase inhibitors are also candidates for cell cycle inhibitors.     

The c-Myc protein induces cell cycle progression and apoptosis through dimerization with Max

The c-Myc protein (Myc) is involved in cellular transformation and mitogenesis, but is also a potent inducer of programmed cell death, or apoptosis. Whether these apparently opposite functions are mediated through common or distinct molecular mechanisms remains unclear. Myc and its partner protein, Max, dimerize and bind DNA in vitro and in vivo through basic/helix-loop-helix/leucine zipper motifs (bHLH-LZ). By using complementary leucine zipper mutants (termed MycEG and MaxEG), which dimerize efficiently with each other but not with their wild-type partners, we demonstrate that both cell cycle progression and apoptosis in nontransformed rodent fibroblasts are induced by Myc-Max dimers. MycEG or MaxEG alone are inactive, but co-expression restores ability to prevent withdrawal from the cell cycle and to induce cell death upon removal of growth factors. Thus, Myc can control two alternative cell fates through dimerization with a single partner, Max.     

Hypoxia arrests ovarian carcinoma cell cycle progression, but invasion is unaffected

Although hypoxic cells are generally resistant to radiation and chemical therapies designed to halt the spread of neoplastic disease, few investigations have been carried out with regard to the molecular mechanisms responsible for this phenomenon. Here, we report of the development of an in vitro model system with which to study the molecular mechanisms involved in the proliferation and invasion of human ovarian carcinoma cells under hypoxia. Results from [(3)]thymidine incorporation experiments indicate that hypoxia triggers cessation of ovarian carcinoma cell DNA synthesis. Flow cytometry analysis of cellular DNA content for hypoxic cultures revealed that cell cycle progression was arrested. This arrest was found to be reversible upon reoxygenation of the cultures. Concomitant with this growth arrest is hypophosphorylation of pRB and a reduction in cyclin A abundance, suggesting that hypoxia induces growth arrest by regulating the activities of these crucial cell cycle-regulatory proteins. In vitro invasion assays revealed that hypoxia has no appreciable effect on the invasive ability of these cells. Immunoblotting established that the detected proteolytic activity was due to the matrix metalloproteinase MMP-2, the M(r) 72,000 type IV collagenase that is most closely associated with the metastatic phenotype in vitro and in vivo. These data support the notion that populations of ovarian carcinoma cells are capable of surviving and invading extracellular matrix during hypoxic conditions and, after a more suitable oxygen environment is reached, giving rise to new cell colonies.     

Ultrastructural aspects of the DNA polymerase alpha distribution during the cell cycle

We studied the nuclear topography of the replicating enzyme DNA polymerase alpha in HeLa cells by transmission electron microscopy and field emission in lens scanning electron microscopy. Cells were synchronized at the G1/S-phase boundary and samples of the different phases of the cell cycle were labeled with an anti-DNA polymerase alpha antibody detected by an immunogold reaction. DNA synthesis was detected by immunogold labeling after bromodeoxyuridine administration. The typical labeling pattern of DNA polymerase alpha observed in G1- and S-phase cells was represented by circular structures 80-100 nm in diameter surrounding an electron-dense area. In double labeled samples these circular structures were associated with bromodeoxyuridine-containing DNA replication sites, forming rosette-like structures. Field emission scanning electron microscopy performed on ultrathin cryosections revealed the chromatin fibers underlying DNA polymerase alpha complexes and showed that the size of the rosette-like structures corresponded to the diameter of chromatin foldings. G2- and M-phase cells showed a spread distribution of DNA polymerase alpha. The evidence of DNA polymerase alpha circular arrangement exclusively in G1- and S-phase cells, obtained by such different approaches, allowed us to consider the three-dimensional structures as DNA replication areas.     

三氧化二砷对T315I突变细胞株KBM5R细胞周期的影响

目的:探讨三氧化二砷(ATO)对T315I突变的伊马替尼(IM)耐药慢性粒细胞白血病(CML)细胞株KBM5R细胞周期的影响,为对抗CML患者的IM耐药性提供理论依据.方法:选择野生型KBM5细胞作为T315I点突变KBM5R细胞的阴性对照组,根据是否经过ATO处理将实验分为KBM5R细胞空白对照组、KBM5R细胞ATO处理组、KBM5细胞空白对照组和KBM5细胞ATO处理组.应用MTT法检测IM和ATO作用后KBM5和KBM5R细胞的增殖活性;流式细胞术检测ATO作用后KBM5和KBM5R细胞周期的变化;Western blotting法检测ATO作用后KBM5和KBM5R细胞周期调节相关蛋白P21和P27表达的变化.结果:IM作用后耐药组KBM5R细胞IC50与野生型KBM5细胞比较明显增高(P<0.01);不同浓度(0.5、1.0、2.0、4.0和8.0 μmol·L-1)ATO于不同时间点(24、48、72和96 h)作用于KBM5和KBM5R细胞,与空白对照组比较,ATO处理组细胞均表现为显著的增殖抑制,且随药物浓度增加或作用时间延长细胞的增殖抑制率显著增加(P<0.05);在相同的药物浓度和时间点,ATO对KBM5R细胞的增殖抑制作用强于野生型KBM5细胞(P<0.05);2.0、4.0和8.0 μmol· L-1 ATO作用细胞48 h后,KBM5和KBM5R细胞周期中G2/M期细胞所占比例均随药物剂量的增加而增加,且相同药物浓度时KBM5R细胞周期中G2/M期细胞所占比例与KBM5细胞比较差异无统计学意义(P>0.05);ATO作用KBM5和KBM5R细胞48 h后,2.0、4.0和8.0 μmol·L-1 ATO处理组与空白对照组比较细胞内P21和P27蛋白表达均增加,且随药物剂量增加而增加.结论:ATO通过上调P21和P27蛋白水平使KBM5R细胞周期阻滞于G2/M期,细胞周期阻滞是ATO抑制T315I点突变细胞株KBM5R增殖的原因之一.