Alterations of cell cycle distribution in the bone marrow of aging mice measured by flow cytometry

The effect of aging on the cell cycle distribution in bone marrow cells was measured by flow cytometry with special reference to the lability in bone marrow physiology. Female C3H mice were examined every 3 h during a 24-h period at the age of 16, 21 and 26 months, vs 3-month-old control mice. Considerable circadian fluctuations were found in the different cell cycle phases in young mice. The rhythmicity patterns were different when comparing different months. In aging mice the variations were dampened, while consistent age-related phase shifts were not seen. The maximal 24-h mean numbers of cells in all three cell phases, but especially the S and G2 phases were reached at 21 months. The relative number of S and G2 phases were significantly reduced in 26-month-old mice, indicating an age-related shift of the proliferative capacity. The present findings are discussed in relation to age-related changes in granulopoiesis and the increase of myelotoxic effects during cancer chemotherapy in aging individuals.     

Cell line-specific differences in the control of cell cycle progression in the absence of mitosis.

This paper reports that there are major differences between mammalian cell lines in the propensity to progress into subsequent cell cycles when mitosis is inhibited with agents that disrupt the assembly of the mitotic spindle apparatus (Colcemid, nocodazole, and taxol). Human HeLa S3 cells, which represent one extreme, remain arrested in mitosis, with elevated levels of cyclin B and p34cdc2 kinase activity. In Chinese hamster ovary cells, at the other extreme, the periodic rise and fall of cyclin B levels and p34cdc2 kinase activity is only transiently inhibited in the absence of mitosis. The cells progress into subsequent cell cycles, without dividing, resulting in serial doublings of cellular DNA content. In general, the propensity to progress into subsequent cell cycles in the absence of mitosis appears to be species related, such that human cell lines remain permanently blocked in a mitotic state, whereas rodent cell lines are only transiently inhibited when spindle assembly is disrupted. We interpret these results to indicate that in mammalian cell lines there exists a checkpoint which serves to couple cell cycle progression to the completion of certain karyokinetic events. Furthermore, either such a checkpoint exists in some cell lines but not in others or the stringency of the control mechanism varies among different cell lines.     

A checkpoint in the cell cycle progression as a therapeutic target to inhibit HIV replication

Human immunodeficiency virus (HIV) expression is boosted after T lymphocyte stimulation. It is not known, however, in which phase(s) of the cell cycle HIV is maximally expressed. We demonstrate here that cell activation induces limited HIV expression and that progression to cell proliferation is required for optimal HIV replication. We also show that the G1/S cell cycle transition is a critical checkpoint in this process and that limiting progression at this step with antiproliferative drugs suppresses HIV replication. These results identify a specific phase of the cell cycle progression that is critical for HIV expression and suggest a new discrete target for anti-HIV treatment.     

Radiation-induced apoptosis and cell cycle progression in TP53-deficient human leukemia cell line HL-60

Human promyelocytic leukemia (HL-60) cells were irradiated with 0.5-100 Gy of gamma radiation and studied for 48 h post irradiation to determine the mode of death and progression of cells through the phases of the cell cycle. HL-60 cells are much more sensitive to radiation-induced loss of clonogenicity (D0 = 2.2 Gy) than to induction of apoptosis at 6 h (D0 for nonapoptotic cells = 32.6 Gy). After doses 20-50 Gy, the onset of massive apoptosis occurred and nonapoptotic cells were in G1/G0 phase of the cell cycle. In contrast, 6 h after irradiation with doses 2.5-10 Gy maximum cells were in S-phase and 16-24 h after irradiation were arrested in G2-phase. Maximum apoptosis occurred 48 h after irradiation with doses 3.5-10 Gy, and cells that died by necrosis were found in 9-44%.     

Hutchinson-Gilford progeria mutant lamin A primarily targets human vascular cells as detected by an anti-Lamin A G608G antibody

Hutchinson-Gilford progeria syndrome (HGPS; Online Mendelian Inheritance in Man accession no. 176670) is a rare disorder that is characterized by segmental premature aging and death between 7 and 20 years of age from severe premature atherosclerosis. Mutations in the LMNA gene are responsible for this syndrome. Approximately 80% of HGPS cases are caused by a G608 (GGC-->GGT) mutation within exon 11 of LMNA, which elicits a deletion of 50 aa near the C terminus of prelamin A. In this article, we present evidence that the mutant lamin A (progerin) accumulates in the nucleus in a cellular age-dependent manner. In human HGPS fibroblast cultures, we observed, concomitantly to nuclear progerin accumulation, severe nuclear envelope deformations and invaginations preventable by farnesyltransferase inhibition. Nuclear alterations affect cell-cycle progression and cell migration and elicit premature senescence. Strikingly, skin biopsy sections from a subject with HGPS showed that the truncated lamin A accumulates primarily in the nuclei of vascular cells. This finding suggests that accumulation of progerin is directly involved in vascular disease in progeria.     

丙戊酸钠对肝癌SMMC-7721细胞增殖和细胞周期的影响及机制

目的:探讨丙戊酸钠(VPA)对人肝癌SMMC-7721细胞增殖、细胞周期及对p21WAF1/CIP1mRNA表达的影响.方法:实验分为空白对照组、PBS组、VPA0.2mmol/L组、VPA1.0mmol/L组和VPA5.0mmol/L组.不同浓度VPA干预人肝癌SMMC-7721细胞24h、48h和72h,采用MTT法检测细胞存活率,流式细胞仪检测细胞周期;干预72h后,用Real-timePCR法检测VPA干预72h后p21WAF1/CIP1mRNA的表达情况.结果:与空白对照组及PBS组比较,不同浓度的VPA作用24h,48h及72h时组肝癌SMMC-7721细胞增殖均出现了不同程度抑制(请将具体数据列出来P<0.05),随着VPA药物浓度升高,细胞增殖抑制作用逐渐增强,随作用时间延长,抑制程度逐渐增强(P<0.05).随药物浓度升高,G1期细胞比例逐渐增多,S期细胞比例逐渐减少,细胞发生G0/G1期阻滞.VPA干预肝癌SMMC-7721细胞72h后,VPA组p21WAF/CIP1mRNA表达较空白对照组及PBS组表达明显升高(请将具体数据列出来P<0.01).结论:VPA可抑制人肝癌SMMC-7721细胞的增殖,且呈时间及剂量依赖性,并诱导出现G0/G1细胞周期阻滞,同时上调p21WAF1/CIP1mRNA的表达.     

A beta-lactone related to lactacystin induces neurite outgrowth in a neuroblastoma cell line and inhibits cell cycle progression in an osteosarcoma cell line.

Lactacystin, a microbial natural product, induces neurite outgrowth in Neuro 2A mouse neuroblastoma cells and inhibits progression of synchronized Neuro 2A cells and MG-63 human osteosarcoma cells beyond the G1 phase of the cell cycle. A related beta-lactone, clasto-lactacystin beta-lactone, formally the product of elimination of N-acetylcysteine from lactacystin, is also active, whereas the corresponding clastolactacystin dihydroxy acid is completely inactive. Structural analogs of lactacystin altered only in the N-acetylcysteine moiety are active, while structural or stereochemical modifications of the gamma-lactam ring or the hydroxyisobutyl group lead to partial or complete loss of activity. The inactive compounds do not antagonize the effects of lactacystin in either neurite outgrowth or cell cycle progression assays. The response to lactacystin involves induction of a predominantly bipolar morphology that is maximal 16-32 h after treatment and is distinct from the response to several other treatments that result in morphological differentiation. Neurite outgrowth in response to lactacystin appears to be dependent upon microtubule assembly, actin polymerization, and de novo protein synthesis. The observed structure-activity relationships suggest that lactacystin and its related beta-lactone may act via acylation of one or more relevant target molecule(s) in the cell.     

表型遗传修饰对人结肠癌细胞周期和抑癌基因表达的调控

目的：分析和探讨同一结肠癌细胞系DNA甲基化和组蛋白乙酰化对抑癌基因表达和细胞周期的影响。方法：培养结肠癌细胞HT－29、SW1116和Colo-320，分别以去甲基化制剂5－氮脱氧胞苷（5－aza-dC）和（或）组蛋白脱乙酰化酶（HDAC）抑制剂trichostatinA(TSA)及丁酸盐干预细胞。提取基因组DNA和RNA，部分行甲基化特异性PCR（MSP）检测p16^INK4A基因启动子区甲基化情况；以RTPCR研究p16^INK4A和p^21WAF1 mRNA表达水平；同时以流式细胞仪分析SW1116和Colo-320细胞周期。结果：干预前，HT－29、SW1116和Colo-320三种结肠癌细胞系中均有较弱的p16^INK4A表达；SW1116和Colo-320细胞的p21^WAF1表达缺如，对于HT－29细胞，1μmol/L的5－aza-dC干预时则无显著改变。在SW1116和Colo-320细胞中，5－aza-dC干预后p16^INK4A表达增强，且以10μmol/L或5μmol/L浓度干预24h者为最明显，相反p21^WAF1仍无明显表达，该两个细胞系经TSA或丁酸盐使细胞阻滞于G1期。结论：三种人结肠癌细胞中，p16^INK4A基因的表达均受甲基化调节。SW1116和Colo-320细胞中p21^WAF1基因表达主要受乙酰化调节，在该两个细胞系中，乙酰化使细胞周期停滞于G1期。     

Thyrotropin prevents apoptosis by promoting cell adhesion and cell cycle progression in FRTL-5 cells

Apoptosis has been shown to be involved in endocrine tissue homeostasis as well as regression due to hormone deprivation. The goal of this study was to induce apoptosis and to investigate a potential role of TSH as a survival factor in thyroid follicular cells (FRTL-5) in vitro. Our results indicated that FRTL-5 cells underwent anchorage-dependent apoptosis when plated in the absence of serum and hormones, but when the cells became attached to the substrate by addition of TSH in the medium, apoptosis was prevented. The apoptosis was evaluated by positive terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling staining, typical apoptotic bodies by electron microscopy, DNA ladder by gel electrophoresis, and subdiploidy by propidium iodide-stained flow cytometry. TSH was shown to prevent apoptosis and maintain cell viability. cAMP partly mimicked this effect, which was inhibited by a specific inhibitor of protein kinase A, H-89. While investigating the mechanisms of apoptosis, we observed that the phosphorylated focal adhesion kinase was strengthened by TSH. Furthermore, FRTL-5 cells were found to undergo growth arrest in the G1 phase in the absence of TSH, accompanied by an elevated level of cyclin-dependent kinase inhibitor, p27, and a decreased level of cyclin D. In contrast, TSH promoted transition from G1 to S phase by decreasing P27 protein and increasing cyclin D expression. We concluded that in addition to regulating growth and differentiation, TSH may function as a survival factor in thyroid cells by preventing anchorage-dependent apoptosis in FRTL-5 cells partly via the cAMP pathway.     

A lamin A protein isoform overexpressed in Hutchinson-Gilford progeria syndrome interferes with mitosis in progeria and normal cells

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder characterized by dramatic premature aging. Classic HGPS is caused by a de novo point mutation in exon 11 (residue 1824, C --> T) of the LMNA gene, activating a cryptic splice donor and resulting in a mutant lamin A (LA) protein termed "progerin/LADelta50" that lacks the normal cleavage site to remove a C-terminal farnesyl group. During interphase, irreversibly farnesylated progerin/LADelta50 anchors to the nuclear membrane and causes characteristic nuclear blebbing. Progerin/LADelta50's localization and behavior during mitosis, however, are completely unknown. Here, we report that progerin/LADelta50 mislocalizes into insoluble cytoplasmic aggregates and membranes during mitosis and causes abnormal chromosome segregation and binucleation. These phenotypes are largely rescued with either farnesyltransferase inhibitors or a farnesylation-incompetent mutant progerin/LADelta50. Furthermore, we demonstrate that small amounts of progerin/LADelta50 exist in normal fibroblasts, and a significant percentage of these progerin/LADelta50-expressing normal cells are binucleated, implicating progerin/LADelta50 as causing similar mitotic defects in the normal aging process. Our findings present evidence of mitotic abnormality in HGPS and may shed light on the general phenomenon of aging.     

Telomere dysfunction and cell cycle checkpoints in hematopoietic stem cell aging

Stem cells are believed to be closely associated with tissue degeneration during aging. Studies of human genetic diseases and gene-targeted animal models have provided evidence that functional decline of telomeres and deregulation of cell cycle checkpoints contribute to the aging process of tissue stem cells. Telomere dysfunction can induce DNA damage response via key cell cycle checkpoints, leading to cellular senescence or apoptosis depending on the tissue type and developmental stage of a specific stem cell compartment. Telomerase mutation and telomere shortening have been observed in a variety of hematological disorders, such as dyskeratosis congenital, aplastic anemia, myelodysplastic syndromes and leukemia, in which the hematopoietic stem cells (HSC) are a major target during the pathogenesis. Moreover, telomere dysfunction is able to induce both cell-intrinsic checkpoints and environmental factors limiting the self-renewal capacity and differentiation potential of HSCs. Crucial components in the cascade of DNA damage response, including ataxia telangiectasia mutated, CHK2, p53, p21 and p16/p19^ARF, play important roles in HSC maintenance and self-renewal in the scenarios of both sufficient telomere reserve and dysfunctional telomere. Therefore, a further understanding of the molecular mechanisms underlying HSC aging may help identity new therapeutic targets for stem cell-based regenerative medicine.     

Adipokines and the insulin resistance syndrome in familial partial lipodystrophy caused by a mutation in lamin A/C

Aims/hypothesis Familial partial lipodystrophy (FPLD) and obesity are both associated with increased risks of type 2 diabetes and cardiovascular disease. Although adipokines have been implicated, few data exist in subjects with FPLD; therefore we investigated a family with FPLD due to a lamin A/C mutation in order to determine how abnormalities of the plasma adipokine profile relate to insulin resistance and the metabolic syndrome. Methods Plasma levels of adiponectin, leptin, resistin, IL-1β, IL-6 and TNF-α in 30 subjects (ten patients, 20 controls) were correlated with indices of metabolic syndrome. Results Compared with controls, FPLD patients had significantly lower plasma levels of adiponectin (3.7±1.0 in FDLP cases vs 7.1±0.72 μg/ml in controls, p=0.02), leptin (1.23±0.4 vs 9.0±1.3 ng/ml, p=0.002) and IL-6 (0.59±0.12 vs 1.04±0.17 pg/ml, p=0.047) and elevated TNF-α (34.8±8.1 vs 13.7±2.7 pg/ml, p=0.028), whereas IL-1β and resistin were unchanged. In both groups, adiponectin levels were inversely correlated with body fat mass (controls, r=−0.44, p=0.036; FDLP, r=−0.67, p=0.025), insulin resistance (controls, r=−0.62, p=0.003; FDLP, r=−0.70, p=0.025) and other features of the metabolic syndrome. TNF-α concentrations were positively related to fat mass (controls, r=0.68, p=0.001; FDLP, r=0.64, p=0.048) and insulin resistance (controls, r=0.86, p=0.001; FDLP, r=0.75, p=0.013). IL-6, IL-1β and resistin did not demonstrate any correlations with the metabolic syndrome in either group. Conclusions/interpretation Low adiponectin and leptin and high TNF-α were identified as the major plasma adipokine abnormalities in FPLD, consistent with the hypothesis that low adiponectin and high TNF-α production may be mechanistically related, and perhaps responsible for the development of insulin resistance and cardiovascular disease in FPLD.     

5-ASA affects cell cycle progression in colorectal cells by reversibly activating a replication checkpoint

BACKGROUND & AIMS: Individuals with inflammatory bowel disease are at risk of developing colorectal cancer (CRC). Epidemiologic, animal, and laboratory studies suggest that 5-amino-salicylic acid (5-ASA) protects from the development of CRC by altering cell cycle progression and by inducing apoptosis. Our previous results indicate that 5-ASA improves replication fidelity in colorectal cells, an effect that is active in reducing mutations. In this study, we hypothesized that 5-ASA restrains cell cycle progression by activating checkpoint pathways in colorectal cell lines, which would prevent tumor development and improve genomic stability. METHODS: CRC cells with different genetic backgrounds such as HT29, HCT116, HCT116(p53-/-), HCT116+chr3, and LoVo were treated with 5-ASA for 2-96 hours. Cell cycle progression, phosphorylation, and DNA binding of cell cycle checkpoint proteins were analyzed. RESULTS: We found that 5-ASA at concentrations between 10 and 40 mmol/L affects cell cycle progression by inducing cells to accumulate in the S phase. This effect was independent of the hMLH1, hMSH2, and p53 status because it was observed to a similar extent in all cell lines under investigation. Moreover, wash-out experiments demonstrated reversibility within 48 hours. Although p53 did not have a causative role, p53 Ser15 was strongly phosphorylated. Proteins involved in the ATM-and-Rad3-related kinase (ATR)-dependent S-phase checkpoint response (Chk1 and Rad17) were also phosphorylated but not ataxia telengectasia mutated kinase. CONCLUSIONS: Our data demonstrate that 5-ASA causes cells to reversibly accumulate in S phase and activate an ATR-dependent checkpoint. The activation of replication checkpoint may slow down DNA replication and improve DNA replication fidelity, which increases the maintenance of genomic stability and counteracts carcinogenesis.