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PubMed2004

Polycomb group gene mel-18 modulates the self-renewal activity and cell cycle status of hematopoietic stem cells.

OBJECTIVE: Mel-18 is a member of the mammalian Polycomb group (PcG) genes. This family of genes regulates global gene expression in many biologic processes, including hematopoiesis and anterior-posterior axis formation by manipulating specific target genes, including members of the Hox family. Here, we demonstrate that mel-18 negatively regulates the self-renewal activity of hematopoietic stem cells (HSCs). MATERIALS AND METHODS: Long-term reconstitution activity was evaluated by competitive repopulating unit (CRU) and mean activity of the stem cells (MAS) assays in vivo in bone marrow cells (BMCs) derived from mel-18(-/-) and mel-18 tg mice. The expression levels of mel-18 and Hoxb4 were measured by quantitative real-time reverse transcription polymerase chain reaction. RESULTS: The Hoxb4 gene was highly expressed in HSCs derived from mel-18(-/-) mice. The observed CRUs were 3.21, 4.77, 3.32, and 1.64 CRU per 10(5) BMCs in mel-18(+/+), mel-18(-/-), C57BL/6, and mel-18 tg, respectively. MAS was 0.58, 0.18, 0.41, and 5.89 in mel-18(+/+), mel-18(-/-), C57BL/6, and mel-18 tg, respectively. The percentage in G0 phase HSCs (lin(-)flk2(-)c-Kit(+)Sca1+ cells) was increased in mel-18(-/-) mice and decreased in mel-18 tg mice. CONCLUSION: Loss or knockdown of mel-18 leads to the expression of Hoxb4, an increase in the proportion of HSCs in G0 phase, and the subsequent promotion of HSC self-renewal. These findings will enable us to develop new approaches for controlling HSC activity for hematopoietic transplantations based on ex vivo expansion of HSCs.

Animals↗

PubMed2003

Gene positional changes relative to the nuclear substructure correlate with the proliferating status of hepatocytes during liver regeneration.

In the interphase nucleus the DNA of higher eukaryotes is organised in loops anchored to a proteinaceous substructure variously named but commonly known as the nuclear matrix. Important processes of nuclear physiology, such as replication, transcription and processing of primary transcripts, occur at macromolecular complexes located at discrete sites upon the nuclear substructure. The topological relationships between gene sequences located in the DNA loops and the nuclear substructure appear to be non-random, thus posing the question of whether such relationships remain invariant or change after the critical nuclear transitions associated with cell proliferation and tissue regeneration in vivo. The hepatocytes are cells that preserve a proliferating capacity that is readily displayed after partial ablation of the liver, leading to liver regeneration in experimental animals such as the rat. Using this animal model coupled to a recently developed PCR-based method for mapping the position of specific DNA sequences relative to the nuclear substructure, we provide evidence that transient changes in the topological relationships between specific genes and the nuclear substructure occur during liver regeneration and that such changes correlate with the actual proliferating status of the cells, thus suggesting that specific transitions in the higher-order DNA structure are characteristic of the quiescent (G0) and replicating (S) phases of the cell cycle in vivo.

Active Transport, Cell Nucleus↗

PubMed2005

The effect of lycopene on cell growth and oxidative DNA damage of Hep3B human hepatoma cells.

Lycopene, the predominant carotenoid in tomatoes and tomato-based foods, is reported to protect against various cancers, especially prostate cancer. We investigated the effect of lycopene on DNA damage and cell growth inhibition in the Hep3B human hepatoma cell line. Lycopene was analyzed by HPLC, and cell proliferation was determined by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. A final lycopene concentration of 0.1-50 microM was added to cells plated in 96-well plates. After a 24-hr incubation, cell viability was measured as absorbance at 570 nm after the MTT assay. The effects of lycopene on cell cycle progression were investigated with flow cytometry. Lycopene induced G0/G1 arrest and S phase block. Oxidative DNA damage was determined by the Comet (single-cell gel electrophoresis) assay. Lycopene inhibited cell growth in a dose-dependent manner. Cell growth was inhibited 20% at 0.2 microM lycopene and 40% at 50 microM lycopene after a 24-hr incubation. In the Comet assay, lycopene-treated cells showed less DNA damage than did placebo-treated cells. The inhibition of Hep3B cell growth in this study demonstrates the antitumor properties of lycopene.

Antioxidants↗

PubMed1995

The tumour suppressor gene product APC blocks cell cycle progression from G0/G1 to S phase.

The APC gene is mutated in familial adenomatous polyposis (FAP) as well as in sporadic colorectal tumours. The product of the APC gene is a 300 kDa cytoplasmic protein associated with the adherence junction protein catenin. Here we show that overexpression of APC blocks serum-induced cell cycle progression from G0/G1 to the S phase. Mutant APCs identified in FAP and/or colorectal tumours were less inhibitory and partially obstructed the activity of the normal APC. The cell-cycle blocking activity of APC was alleviated by the overexpression of cyclin E/CDK2 or cyclin D1/CDK4. Consistent with this result, kinase activity of CDK2 was significantly down-regulated in cells overexpressing APC although its synthesis remained unchanged, while CDK4 activity was barely affected. These results suggest that APC may play a role in the regulation of the cell cycle by negatively modulating the activity of cyclin-CDK complexes.

3T3 Cells↗

PubMed2000

Combined cytotoxic action of paclitaxel and ceramide against the human Tu138 head and neck squamous carcinoma cell line.

PURPOSE: Paclitaxel, a chemotherapeutic agent used in the treatment of recalcitrant ovarian and breast as well as other neoplasms, is being investigated for the treatment of squamous cell carcinoma of the head and neck. Our previous studies have demonstrated that exogenously added ceramide enhances apoptosis in paclitaxel-exposed human leukemic cells. In this study, we showed that exogenous ceramide augmented paclitaxel-induced apoptosis in Tu138 cells in vitro when added simultaneously in combination with the paclitaxel. METHODS: The combined cytotoxic effects of paclitaxel and ceramide exposure against Tu138 cells were assessed by an MTT dye assay, cell cycle analysis, TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay, and isobologram analysis for synergistic activity. RESULTS: The MTT dye assay results indicated augmentation of time- and concentration-dependent paclitaxel-mediated cell cytotoxicity by simultaneous ceramide treatment. Paclitaxel treatment of Tu138 cells also resulted in an accumulation of cells in the G2-M phase of the cell cycle. This paclitaxel-mediated G2-M phase accumulation decreased significantly with the addition of ceramide, indicating that combined paclitaxel/ceramide treatment resulted in the elimination of Tu138 cells from the S and/or G2-M phases of the cell cycle. Furthermore, ceramide enhancement of paclitaxel-mediated apoptosis was also detected by the TUNEL assay. CONCLUSION: Our results suggest that paclitaxel/ceramide combination therapy may be an attractive alternative to conventional methods of chemotherapy for head and neck cancer, and should be further explored.

Carcinoma, Squamous Cell↗

PubMed2006

Cigarette tar phenols impede T cell cycle progression by inhibiting cyclin-dependent kinases.

Cigarette smoking causes profound suppression of pulmonary T cell responses, which is associated with increased susceptibility to respiratory tract infections and decreased tumor surveillance. We previously demonstrated that the phenolic compounds in cigarette tar inhibit blastogenesis and interfere with human T cell cycle progression. To identify the mechanism by which cell cycle arrest occurs, we examined the effects of these compounds on cyclin-dependent kinases (Cdk) that control the G0/G1 transition. We found that hydroquinone inhibited induction of Cdk4 and Cdk6 kinase activities by >80%, while catechol and phenol were markedly less potent. HQ did not affect mitogenic induction of the Cdk6 protein, but inhibited expression of cyclin D3 by >90% resulting in a dramatic reduction in proper Cdk6/Cyclin D3 complex formation.

Cell Cycle↗

PubMed2002

Flow cytometric cell cycle analysis of somatic cells primary cultures established for bovine cloning.

An important factor governing developmental rates of somatic cloned embryos is the phase of the cell cycle of donor nuclei. The aim of this experiment was to investigate the distribution of cell cycle phases in bovine cumulus and fibroblast cells cultured using routine treatment, and under cell cycle-arresting treatments. The highest percentages of cumulus cells in the G0 + G1 stage were observed in uncultured, frozen/thawed cells originating from immature oocytes (79.8 +/- 2.2%), fresh and frozen/thawed cells from in vitro matured oocytes (84.1 +/- 6.2 and 77.8 +/- 5.7%, respectively), and in cycling cells (72.7 +/- 16.3 and 78.4 +/- 11.2%, respectively for cumulus cells from immature and in vitro matured oocytes). Serum starvation of cumulus cultures markedly decreased percentages of cells in G0 + G1, and prolonged starvation significantly increased (P < 0.05) percentages of cells in G2 + M phase. Culture of cumulus cells to confluency did not increase percentages of cells in G0 + G1. Contrary to findings in cumulus cells, significantly higher percentages of cells in G0 + G1 were apparent when fibroblast cells were cultured to confluency or serum starved, and significantly increased (P < 0.01) as the starvation period was prolonged. It is concluded that for particular cell types specific strategies should be used to attain improvements in the efficiency of cloning procedures.

Animals↗

PubMed1998

Levels of polyadenylation factor CstF-64 control IgM heavy chain mRNA accumulation and other events associated with B cell differentiation.

Cleavage stimulation factor (CstF) is one of the multiple factors required for mRNA polyadenylation. The concentration of one CstF subunit (CstF-64) increases during activation of B cells, and this is sufficient to switch IgM heavy chain mRNA expression from membrane-bound form to secreted form. To extend this observation, we disrupted the endogenous CstF-64 gene in the B cell line DT40 and replaced it with a regulatable transgene. Strikingly, a 10-fold decrease in CstF-64 concentration did not markedly affect cell growth but specifically and dramatically reduced accumulation of IgM heavy chain mRNA. Further reduction caused reversible cell cycle arrest in G0/G1 phase, while depletion resulted in apoptotic cell death. Our results indicate that CstF-64 plays unexpected roles in regulating gene expression and cell growth in B cells.

Animals↗

PubMed2003

Prolonged exposure to high glucose impaired cellular behavior of normal human corneal epithelial cells.

PURPOSE: To determine the effect of prolonged exposure to high glucose on cellular behavior of normal human corneal epithelial cells (HCEC). METHODS: HCEC were cultured in medium under normal or high glucose conditions for 14 days. Proliferation was evaluated by direct cell counting and [(3)H]thymidine incorporation. Cell cycle analysis was performed using flow cytometry. The ability of HCEC to attach to type I collagen was evaluated using a short-term colorimetric adhesion assay. The effect of high glucose on the expression of integrin alpha(3)beta(1) was also evaluated using flow cytometry. RESULTS: Cell number and [(3)H]thymidine incorporation under high glucose conditions decreased compared with those under normal glucose conditions. The cells exposed to high glucose were G(0)/G(1) than untreated cells. The adhesion ability of HCEC under high glucose conditions decreased compared to normal glucose conditions. Expression of integrin alpha( 3)beta(1) was down-regulated under high glucose conditions. CONCLUSIONS: High glucose had deleterious effects on cellular behavior of HCEC, which might cause delayed corneal epithelial wound healing in diabetic keratopathy.

Antibodies↗

PubMed2004

Allyl-isothiocyanate causes mitotic block, loss of cell adhesion and disrupted cytoskeletal structure in HT29 cells.

Epidemiological evidence indicates that Brassica vegetables protect against colorectal cancer. Brassicas contain glucosinolates, the breakdown products of which exert antiproliferative effects against cancer cells. We have examined the effects of allyl-isothiocyanate (AITC), a major breakdown product of the glucosinolate sinigrin, on proliferation and death of colorectal cancer cells. HT-29 colorectal cells were exposed to AITC for 24 h and the number of adherent and detached cells determined. Both populations were analysed for cell-cycle characteristics and examined by light and electron microscopy for features of apoptosis and mitosis. Evidence of apoptosis was also determined by flow cytometric analysis of Annexin V staining in the detached population of cells. AITC-treated cells were also stained for alpha-tubulin. Treatment caused cells to round up after 7 h of exposure and subsequently detach. At 24 h these cells were blocked in mitosis. Detached AITC-treated cells showed no signs of apoptosis as assessed by morphological features or by Annexin V staining but they did show evidence of disrupted tubulin. AITC inhibits proliferation of cancer cells by causing mitotic block associated with disruption of alpha-tubulin in a manner analogous to a number of chemotherapeutic agents.

Annexin A5↗

PubMed2003

NC381, a novel anticancer agent, arrests the cell cycle in G0-G1 and inhibits lung tumor cell growth in vitro and in vivo.

Although clotrimazole (CLT), an antifungal drug, inhibits tumor cell proliferation and angiogenesis, its clinical application is hampered by significant hepatotoxicity due to the presence of an imidazole moiety. In our attempts to develop CLT analogs that are devoid of imidazole and are as efficacious as CLT, one pharmacophore designated NC381 was generated and shown to inhibit tumor cell growth via a mechanism similar to that of CLT. In vitro, treatment of NCI-H460 nonsmall cell lung cancer (NSCLC) cells with NC381 inhibited growth in a time-dependent manner. Flow cytometric analysis demonstrated that the decrease in cell growth was associated with inhibition of cell cycle progression at the G(1)-S phase transition, resulting in G(0)-G(1) arrest. There was a concomitant inhibition of cyclin D1 expression and subsequent reduction in the formation of the cyclin D1-CDK4 complex. Consistent with a decrease in the cyclin D1-CDK4 complex, NC381 treatment resulted in significant inhibition of pRb phosphorylation. There also were changes in the activity of cell cycle-related proteins, including p16(Ink4) and p27(Kip1). Together, these results are consistent with a model in which NC381 arrests cell cycle progression via inhibition of the pathway that promotes exit from the G(1) phase of the cell cycle. Furthermore, the clinical applicability of NC381 was evaluated in an in vivo murine xenograft model of human NSCLC (NCI-H460). NC381 treatment resulted in significant inhibition of tumor growth. Given the poor prognosis and the limited treatment options available, the present results underscore the potential of NC381 in the treatment of human NSCLC.

Animals↗

PubMed2006

Peroxisome proliferator-activated receptor-gamma activates p53 gene promoter binding to the nuclear factor-kappaB sequence in human MCF7 breast cancer cells.

The aim of the present study was to provide new mechanistic insight into the growth arrest and apoptosis elicited by peroxisome proliferator-activated receptor (PPAR)gamma in breast cancer cells. We ascertained that PPARgamma mediates the inhibition of cycle progression in MCF7 cells exerted by the specific PPARgamma agonist rosiglitazone [BRL4653 (BRL)], because this response was no longer notable in the presence of the receptor antagonist GW9662. We also provided evidence that BRL is able to up-regulate mRNA and protein levels of the tumor suppressor gene p53 and its effector p21(WAF1/Cip1) in a time- and dose-dependent manner. Moreover, in transfection experiments with deletion mutants of the p53 gene promoter, we documented that the nuclear factor-kappaB sequence is required for the transcriptional response to BRL. Interestingly, EMSA showed that PPARgamma binds directly to the nuclear factor-kappaB site located in the promoter region of p53, and chromatin immunoprecipitation experiments demonstrated that BRL increases the recruitment of PPARgamma on the p53 promoter sequence. Next, both PPARgamma and p53 were involved in the cleavage of caspases-9 and DNA fragmentation induced by BRL, given that GW9662 and an expression vector for p53 antisense blunted these effects. Our findings provide evidence that the PPARgamma agonist BRL promotes the growth arrest and apoptosis in MCF7 cells, at least in part, through a cross talk between p53 and PPARgamma, which may be considered an additional target for novel therapeutic interventions in breast cancer patients.

Base Pairing↗

PubMed2006

[Mechanism of cell proliferation--cell cycle, oncogenes, and senescence].

Cell proliferation is regulated through a transition between the G0 phase and cell cycle. We isolated a mammalian temperature-sensitive mutant cell line defective in the function from the G0 phase to cell cycle. Senescent human somatic cells fail to enter into the cell cycle from the G0 phase with stimulation by any growth factor. Telomere shortening was found to be a cause of cellular senescence, and reexpression of telomerase immortalized human somatic cells. Immortalized human somatic cells showed normal phenotypes and were useful not only for basic research but also for clinical and applied fields. The importance of p53 and p21 activation/induction i now well accepted in the signal transduction process from telomere shortening to growth arrest, but the precise mechanism is largely unknown as yet. We found that the MAP kinase cascade and histone acetylase have an important role in the signaling process to express p21. Tumor tissues and cells were found to have strong telomerase activity, while most normal somatic human tissues showed very weak or no activity. Telomerase activity was shown to be a good marker for early tumor diagnosis because significant telomerase activity was detected in very early tumors or even in some precancerous tissues compared with adjacent normal tissues. Telomere/telomerase is a candidate target for cancer chemotherapeutics, and an agent that abrogated telomere functions was found to kill tumor cells effectively by inducing apoptosis whereas it showed no effect on the viability of normal cells.

Animals↗

PubMed1998

All-trans retinoic acid converts E2F into a transcriptional suppressor and inhibits the growth of normal human bronchial epithelial cells through a retinoic acid receptor- dependent signaling pathway.

Retinoids, including retinol and retinoic acid derivatives, maintain the normal growth and differentiation of human bronchial epithelial (HBE) cells and are under investigation as agents for lung cancer prevention. In this study, we examined the biologic effects of retinoids on normal HBE cells and the molecular mechanisms of retinoid actions. At a dose of 10(-6) M, all-trans retinoic acid (t-RA) suppressed the proliferation of normal HBE cells, which accumulated in the G0 phase. No evidence of programmed cell death was observed. The class of retinoid nuclear receptor that mediated the growth arrest was explored. Normal HBE cell growth was suppressed by a retinoid that selectively activates retinoic acid receptors but not by one that activates retinoid X receptors. The E2F transcription factor has demonstrated a role in G0 entry through transcriptional suppression of genes that induce cell cycle progression. To investigate the role of E2F in retinoid signaling, transient transfection assays were performed using reporter plasmids containing E2F-binding sites. Findings from these experiments suggested that t-RA treatment converted E2F into a transcriptional suppressor. Supporting this possibility, t-RA inhibited the expression of the E2F target genes B-myb, cyclin A, and cyclin E. Further, t-RA increased the levels of nuclear E2F-4, p107, and p130 and enhanced the binding of E2F-4 to p107, which have been associated with the conversion of E2F into a transcriptional suppressor in other cells. These findings point to retinoic acid receptor- and E2F-dependent pathways as potential mediators of retinoid-induced growth arrest in normal HBE cells and have implications for the use of retinoids in clinical trials on the prevention of lung cancer.

Apoptosis↗

PubMed1993

Molecular cloning of a diverged homeobox gene that is rapidly down-regulated during the G0/G1 transition in vascular smooth muscle cells.

Adult vascular smooth muscle cells dedifferentiate and reenter the cell cycle in response to growth factor stimulation. Here we describe the molecular cloning from vascular smooth muscle, the structure, and the chromosomal location of a diverged homeobox gene, Gax, whose expression is largely confined to the cardiovascular tissues of the adult. In quiescent adult rat vascular smooth muscle cells, Gax mRNA levels are down-regulated as much as 15-fold within 2 h when these cells are induced to proliferate with platelet-derived growth factor (PDGF) or serum growth factors. This reduction in Gax mRNA is transient, with levels beginning to rise between 8 and 24 h after mitogen stimulation and returning to near normal by 24 to 48 h. The Gax down-regulation is dose dependent and can be correlated with the mitogen's ability to stimulate DNA synthesis. PDGF-AA, a weak mitogen for rat vascular smooth muscle cells, did not affect Gax transcript levels, while PDGF-AB and -BB, potent mitogens for these cells, were nearly as effective as fetal bovine serum. The removal of serum from growing cells induced Gax expression fivefold within 24 h. These data suggest that Gax is likely to have a regulatory function in the G0-to-G1 transition of the cell cycle in vascular smooth muscle cells.

Animals↗

PubMed1999

The involvement of chromatin condensation in camptothecin-induced chromosome breaks in G0 human lymphocytes.

In the present study we evaluated campthotecin (CPT)-induced chromosomal damage in human lymphocytes in the G0 phase of the cell cycle as revealed by the premature chromosome condensation technique. The results obtained here indicate that CPT was able to induce chromosome fragments in the G0 phase of the cell cycle of human lymphocytes as detected in prematurely condensed chromosomes. This result appears to be rather surprising, since the DNA lesions produced by CPT (e.g. 'protein concealed' DNA single-strand breaks) should not produce any damage in G0. A possible explanation for this result could come from much evidence to suggest that chromatin condensation processes are significantly involved in the conversion of DNA lesions into chromosome breaks in prematurely condensed chromosomes. The unexpected clastogenic behaviour of CPT can be explained taking into account the chromosome condensation induced by mitosis promoting factors when human lymphocytes are fused in G0, thus converting the 'protein concealed' DNA single-strand breaks induced by CPT into chromosome breaks. The same perspective should be taken into consideration for breaks induced by CPT under normal physiological conditions in the G2 phase of the cell cycle.

Animals↗

PubMed1997

The subcellular localization of E2F-4 is cell-cycle dependent.

The E2F family of transcription factors plays a crucial role in cell cycle progression. E2F activity is tightly regulated by a number of mechanisms, which include the timely synthesis and degradation of E2F, interaction with retinoblastoma protein family members ("pocket proteins"), association with DP heterodimeric partner proteins, and phosphorylation of the E2F/DP complex. Here we report that another mechanism, subcellular localization, is important for the regulation of E2F activity. Unlike E2F-1, -2, or -3, which are constitutively nuclear, ectopic E2F-4 and -5 were predominantly cytoplasmic. Cotransfection of expression vectors encoding p107, p130, or DP-2, but not DP-1, resulted in the nuclear localization of E2F-4 and -5. Moreover, the transcriptional activity of E2F-4 was markedly enhanced when it was invariably nuclear. Conversely, it was reduced when the protein was excluded from the nucleus, implying that E2F-4 transcription function depends upon its cytological location. In keeping with this, the nuclear/cytoplasmic ratios of endogenous E2F-4 changed as cells exited G0, with high ratios in G0 and early G1 and a progressive increase in cytoplasmic E2F-4 as cells approached S phase. Thus, the subcellular location of E2F-4 is regulated in a cell cycle-dependent manner, providing another potential mechanism for its functional regulation.

3T3 Cells↗

PubMed1992

A cryptopromoter is activated in the proliferating cell nuclear antigen gene of growth arrested cells.

We have examined the regulation of the proliferating cell nuclear antigen gene (PCNA) in a hamster fibroblast cell line (tk-ts13) which is temperature sensitive for growth. These tk-ts13 cells, at the restrictive temperature, are growth arrested in the G1 phase of the cell cycle. The cells were stably transfected with a full length human PCNA gene, and the resulting cell lines (K525 cells) were analyzed. We find that, in growth arrested K525 cells, a cryptopromoter is activated in the transfected human PCNA gene. The cryptopromoter resides in intron 4 which is necessary for proper regulation of the PCNA gene. Removal of this intron leads to increased expression of PCNA in cells which have entered the G0 state. An Alu sequence residing in intron 4 is implicated as the promoter element which is active during growth arrest.

Animals↗