Senescence-like changes induced by expression of p21~(Waf1/Cip1) in NIH3T3 cell line

P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/Cip1 involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21Waf1/Cip1 and cellular senescence. While in murine cells, the role of p21Waf1/Cip1 is indefinite. We explored this issue using NIH3T3 cells with inducible p21Waf1/Cip1 expression. Induction of p21Waf1/Cip1 triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21Waf1/Cip1-transduced NIH3T3 cells expressed β-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p21Waf1/Cip1 can also induce senescence-like changes in murine cells.     

Senescence-like changes induced by expression of p21Waf1/Cip1 in NIH3T3 cell line

P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/cip1involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidencefor a link between p21Waf1/cip1 and cellular senescence. While in murine cells, the role of p21Waf1/Cip1is indefinite. We explored this issue using NIH3T3 cells with inducible p21Waf1/cip1 expression. Induc-tion of p21Waf1/Cip1 triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features,such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed thatp21Waf1/Cip1-transduced NIH3T3 cells expressedβ-galactosidase activity at pH 6.0, which is known to bea marker of senescence. Our results suggest that p21Waf1/cipx can also induce senescence-like changes inmurine cells.     

Indoleamine 2, 3-dioxygenase （IDO） is essential for dendritic cell activation and chemotactic responsiveness to chemokines

Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fetal alloantigens during murine pregnancy. In mice, IDO expression is an inducible feature of specific subsets of dendritic cells (DCs),and is important for T cell regulatory properties. However, the effect of IDO and tryptophan deprivation on DC functions remains unknown. We report here that when tryptophan utilization was prevented by a pharmacological inhibitor of IDO, 1-methyl tryptophan (1MT), DC activation induced by pathogenic stimulus lipopolysaccharide (LPS) or inflammatory cytokine TNF-α was inhibited both phenotypically and functionally. Such an effect was less remarkable when DC was stimulated by a physiological stimulus, CD40 ligand. Tryptophan deprivation during DC activation also regulated the expression of CCR5 and CXCR4, as well as DC responsiveness to chemokines. These results suggest that tryptophan usage in the microenvironment is essential for DC maturation, and may also play a role in the regulation of DC migratory behaviors.     

A glucocorticoid amplifies IL-2-induced selective expansion of CD4^＋CD25^＋FOXP3^＋ regulatory T cells in vivo and suppresses graft-versus-host disease after allogeneic lymphocyte transplantation

Regulatory T （Treg） cells are a subpopulation of T cells that not only prevent autoimmunity, but also control a wide range of T cell-dependent immune responses. Glucocorticoid treatment （dexamethasone, or Dex） has been reported to amplify IL-2-mediated selective in vivo expansion of Treg cells. We simultaneously administered Dex and IL-2 to the donor in a murine allogeneic lymphocyte transplantation model to expand functional suppressive CD4＋CD25＋FOXP3＋ T cells in the graft and to raise the regulatory T cell/effector T cell （Treg/ Teff） ratio to prevent graft-versus-host disease （GVHD）. After combined treatment of the donor with Dex （5 mg/kg/day） and IL-2 （300,000 IU/mouse/day） for 3 days, grafts were subjected to flow cytometric analysis, and transplantation was carried out from male C57BL/6 mice to female BALB/c mice aged 8-12 weeks. Results showed that short-term simultaneous administration of Dex and IL-2 markedly expanded functional suppressive CD4＋CD25＋FOXP3＋ T cells in the murine spleen. In this murine allogeneic transplantation model, the grafts from donors with Dex and IL-2 pre-treatment led to a longer survival time for the recipients than for the control group （median survival time 〉 60 day vs. 12 day, P = 0.0002）. The ratio of Treg/Teff also increased remarkably （0.43 ±0.15 vs. 0.14 ± 0.01, P = 0.01）. This study demonstrated that co-stimulation with Dex and IL-2 selectively expanded functional CD4＋CD25＋FOXP3＋ T cells in vivo, and that grafts from donors pre-treated with Dex and IL-2 led to longer survival time and greater suppression of GVHD after allogeneic transplantation. Thus, GVHD can be suppressed by the specific expansion of regulatory T cells with Dex and IL-2 in graft donors.     

Development of cancer-initiating cells and immortalized cells with genomic instability

Cancers that develop after middle age usually exhibit genomic instability and multiple mutations. This is in direct contrast to pediatric tumors that usually develop as a result of specific chromosomal translocations andepigenetic aberrations. The development of genomic instability is associated with mutations that contribute to cellular immortalization and transformation. Cancer occurs when cancer-initiating cells(CICs), also called cancer stem cells, develop as a result of these mutations. In this paper, we explore how CICs develop as a result of genomic instability, including looking at which cancer suppression mechanisms are abrogated. A recent in vitro study revealed the existence of a CIC induction pathway in differentiating stem cells. Under aberrant differentiation conditions, cells become senescent and develop genomic instabilities that lead to the development of CICs. The resulting CICs contain a mutation in the alternative reading frame of CDKN2A(ARF)/p53 module, i.e., in either ARF or p53. We summarize recently established knowledge of CIC development and cellular immortality, explore the role of the ARF/p53 module in protecting cells from transformation, and describe a risk factor for genomic destabilization that increases during the process of normal cell growth and differentiation and is associated with the downregulation of histone H2 AX to levels representative of growth arrest in normal cells.     

Viral infections and cell cycle G2／M regulation

Progression of cells from G2 phase of the cell cycle to mitosis is a tightly regulated cellular process that requires activation of the Cdc2 kinase, which determines onset of mitosis in all eukaryotic cells. In both human and fission yeast(Schizosaccharomyces pombe) cells, the activity of Cdc2 is regulated in part by the phosphorylation status of tyrosine 15 (Tyrl5) on Cdc2, which is phosphorylated by Weel kinase during late G2 and is rapidly dephosphorylated by the Cdc25 tyrosine phosphatase to trigger entry into mitosis. These Cdc2 regulators are the downstream targets of two wellcharacterized G2/M checkpoint pathways which prevent cells from entering mitosis when cellular DNA is damaged or when DNA replication is inhibited. Increasing evidence suggests that Cdc2 is also commonly targeted by viral proteins,which modulate host cell cycle machinery to benefit viral survival or replication. In this review, we describe the effect of viral protein R (Vpr) encoded by human immunodeficiency virus type 1 (HIV-Ⅰ) on cell cycle G2/M regulation. Based on our current knowledge about this viral effect, we hypothesize that Vpr induces cell cycle G2 arrest through a mechanism that is to some extent different from the classic G2/M checkpoints. One the unique features distinguishing Vpr-induced G2 arrest from the classic checkpoints is the role of phosphatase 2A (PP2A) in Vpr-induced G2 arrest.Interestingly, PP2A is targeted by a number of other viral proteins including SV40 small T antigen, polyomavirus T antigen, HTLV Tax and adenovirus E4orf4. Thus an in-depth understanding of the molecular mechanisms underlying Vpr-induced G2 arrest will provide additional insights into the basic biology of cell cycle G2/M regulation and into the biological significance of this effect during host-pathogen interactions.     

Cell transformation as aberrant differentiation：Environmentally dependent spontaneous transformation of NIH 3T3 cells

NIH 3T3 cells, a mouse fibroblast cell line used as routine target cells for transfection experiments, undergo spontaneous transformation in our experiments after they form a confluent sheet in medium containing fetal bovine serum (FBS) or lower coneentration of calf serum (CS). The transformation takes the form of foci of multiplying cells among the surrounding cells which have stopped cell division. However, no focus of transformed cells could be seen in medium containing high concentration (10％) of CS. Further experiments indicated that the frequency of transformation is highly dependent on the concentration of serum and the transformation in CS is changeable when the cells are passaged in FBS. 3~H-thymidine autoradiography has been proved to be a sensitive measurement indicator for focus formation. Our results suggest that the high frequency of transformation and its dependence on confluency as well as on medium composition are characteristics of cell differentiation rather than mutation. The role of the NIH 3T3 cell line as a cancer-initiated cell population and its accelerated transformation by ras oncogene might be considered as a form of tumor promotion is discussed.     

Fucoxanthin induces growth arrest and apoptosis in human bladder cancer T24 cells by up-regulation of p21 and down-regulation of mortalin

Fucoxanthin, a natural carotenoid, has been reported to have anti-cancer activity in human colon cancer cells, human prostate cancer cells, human leukemia cells, and human epithelial cervical cancer cells. This study was undertaken to evaluate the molecular mechanisms of fuco- xanthin against human bladder cancer T24 cell line. MTT analysis results showed that 5 and 10 ixM fucoxanthin inhibited the proliferation of T24 cells in a dose- and time- dependent manner accompanied by the growth arrest at Go/G1 phase of cell cycle, which is mediated by the up-regu- lation of p21, a cyclin-dependent kinase （CDK）-inhibitory protein and the down-regulation of CDK-2, CDK-4, cyclin D1, and cyclin E. In addition, 20 and 40 μM fucoxanthin induced apoptosis of T24 cells by the abrogation of morta- lin-p53 complex and the reactivation of nuclear mutant- type p53, which also had tumor suppressor function as wild-type p53. All these results demonstrated that the anti- cancer activity of fucoxanthin on T24 cells was associated with cell cycle arrest at Go/G1 phase by up-regulation of p21 at low doses and apoptosis via decrease in the expres- sion level of mortalin, which is a stress regulator and a mem- ber of heat shock protein 70, followed by up-regulation of cleaved caspase-3 at high doses.     

Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ inter- acted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.     

Altered gene expression profiles of NIH3T3 cells regulated by human lung cancer associated gene CT120

CT120, a novel membrane-associated gene implicated in lung carcinogenesis, was previously identified from chromosome 17pl 3.3 locus, a hot mutation spot involved in human malignancies. In the present study, we further determined that CT120 ectopic expression could promote cell proliferation activity of NIH3T3 cells using MTS assay, and monitored the downstream effects of CT120 in NIH3T3 cells with Atlas mouse cDNA expression arrays. Among 588 known genes, 133 genes were found to be upregulated or downregulated by CT120. Two major signaling pathways involved in cell proliferation, cell survival and anti-apoptosis were overexpressed and activated in response to CT120:One is the Raf/MEK/Erk signal cascades and the other is the PI3K/Akt signal cascades, suggesting that CT120 might contribute, at least in part, to the constitutively activation of Erk and Akt in human lung caner cells. In addition, some tumor metastasis associated genes cathepsin B, cathepsin D, cathepsin L, MMP-2/TIMP-2 were also upregulated by CT120, upon which CT120 might be involved in tumor invasiveness and metastasis. In addition, CT120 might play an important role in tumor progression through modulating the expression of some candidate “Lung Tumor Progression”genes including B-Raf, Rab-2, BAX, BAG-1, YB-1, and Cdc42.     

Intracellular levels of calmodulin are increased in transformed cells

By using Hoechst 33342,rabbit anti calmodulin antibody,FITC-labeled goat anti rabbit IgG and SR101(sulfo rhodamine 101)simultaneously to stain individual normal and transformed cells,the microspectrophotometric analysis demonstrated that 3 markers which represented the nucleus,calmodulin and total protein respectively,could be recognized in individualj cells without interference,The phase of the cell cycle was determined by DNA content(Hoechst 33342),We found that in transformed cells(NIH3T3) tsRSV-LA90,cultured at 33℃ and transformed C3H10T1/2 Cells),the ration of calmodulin to total protein (based on the phases of cell cycle)was higher than that in normal cells (NIH3T3 tsRSV-LA90 cells,cultured at 39℃ and C3H10T1/2 cells)in every cell cycle phase,This ration increased obviously only from G1 to S phase in either normal or transformed cells.The results showed that calmodulinreally increased during the transformation,and its increase was specific.In the meantime when cells proceeded from G1 to S.the intraceollular calmodulin content also increased specifically.     

Downregulation of CD4＋CD25＋ regulatory T cells may underlie enhanced Th1 immunity caused by immunization with activated autologous T cells

Regulatory T cells （Treg） play important roles in immune system homeostasis, and may also be involved in tumor immunotolerance by suppressing Th1 immune response which is involved in anti-tumor immunity. We have previously reported that immunization with attenuated activated autologous T cells leads to enhanced anti-tumor immunity and upregulated Thl responses in vivo. However, the underlying molecular mechanisms are not well understood. Here we show that Treg function was significantly downregulated in mice that received immunization of attenuated activated autologous T cells. We found that Foxp3 expression decreased in CD4＋CD25＋ T cells from the immunized mice. Moreover, CD4＋CD25＋Foxp3＋ Treg obtained from immunized mice exhibited diminished immunosuppression ability compared to those from naive mice. Further analysis showed that the serum of immunized mice contains a high level ofanti-CD25 antibody （about 30 ng/ml, p〈0.01 vs controls）. Consistent with a role ofanti-CD25 response in the downregulation of Treg, adoptive transfer of serum from immunized mice to naive mice led to a significant decrease in Treg population and function in recipient mice. The triggering of anti-CD25 response in immunized mice can be explained by the fact that CD25 was induced to a high level in the ConA activated autologous T cells used for immunization. Our results demonstrate for the first time that immunization with attenuated activated autologous T cells evokes anti-CD25 antibody production, which leads to impeded CD4＋CD25＋Foxp3＋ Treg expansion and function in vivo. We suggest that dampened Treg function likely contributes to enhanced Thl response in immunized mice and is at least part of the mechanism underlying the boosted anti-tumor immunity.     

Homotypic Vacuole Fusion Requires VTI11 and Is Regulated by Phosphoinositides

Most plant cells contain a large central vacuole that is essential to maintain cellular turgor. We report a new mutant allele of VTI11 that implicates the SNARE protein VTI11 in homotypic fusion of protein storage and lytic vacuoles. Fusion of the multiple vacuoles present in vtill mutants could be induced by treatment with Wortmannin and LY294002, which are inhibitors of Phosphatidylinositol 3-Kinase （PI3K）. We provide evidence that Phosphatidylinositol 3-Phosphate （Ptdlns（3）P） regulates vacuole fusion in vtill mutants, and that fusion of these vacuoles requires intact microtubules and actin filaments. Finally, we show that Wortmannin also induced the fusion of guard cell vacuoles in fava beans, where vacuoles are naturally fragmented after ABA-induced stomata closure. These results suggest a ubiquitous role of phosphoinositides in vacuole fusion, both during the development of the large central vacuole and during the dynamic vacuole remodeling that occurs as part of stomata movements.     

CHK2 kinase in the DNA damage response and beyond

The serine/threonine kinase CHK2 is a keycomponent of the DNA damage response. In human cells, following genotoxic stress, CHK2 is activated and phosphorylates 〉20 proteins to induce the appropriate cellular response, which, depending on the extent of damage, the cell type, and other factors, could be cell cycle checkpoint activation, induction of apoptosis or senescence, DNA repair, or tolerance of the damage. Recently, CHK2 has also been found to have cellular functions independent of the presence of nuclear DNA lesions. In par- ticular, CHK2 participates in several molecular processes involved in DNA structure modification and cell cycle progression. In this review, we discuss the activity of CHK2 in response to DNA damage and in the maintenance of the biological functions in unstressed cells. These activities are also considered in relation to a possible role of CHK2 in tumorigenesis and, as a consequence, as a target of cancer therapy.     

Downregulation of wild-type p53 protein by HER-2／neu mediated PI3K pathway activation in human breast cancer cells： its effect on cell proliferation and implication for therapy

Overexpression and activation of HER-2/neu (also known as c-erbB-2), a proto-oncogene, was found in about 30% of human breast cancers, promoting cancer growth and making cancer cells resistant to chemo- and radio-therapy.Wild-type p53 is crucial in regulating cell growth and apoptosis and is found to be mutated or deleted in 60-70% of human cancers. And some cancers with a wild-type p53 do not have normal p53 function, suggesting that it is implicated in a complex process regulated by many factors. In the present study, we showed that the overexpression of HER-2/neu could decrease the amount of wild-type p53 protein via activating PI3K pathway, as well as inducing MDM2 nuclear translocation in MCF7 human breast cancer cells. Blockage of PI3K pathway with its specific inhibitor LY294002 caused G1-S phase arrest, decreased cell growth rate and increased chemo- and radio-therapeutic sensitivity in MCF7 cells expressing wild-type p53. However, it did not increase the sensitivity to adriamycin in MDA-MB-453 breast cancer cells containing mutant p53. Our study indicates that blocking PI3K pathway activation mediated by HER-2/neu overexpression may be useful in the treatment of breast tumors with HER-2/neu overexpression and wild-type p53.     

Blocking the Metabolism of Starch Breakdown Products in Arabidopsis Leaves Triggers Chloroplast Degradation

In most plants, a large fraction of photo-assimilated carbon is stored in the chloroplasts during the day as starch and remobilized during the subsequent night to support metabolism. Mutations blocking either starch synthesis or starch breakdown in Arabidopsis thaliana reduce plant growth. Maltose is the major product of starch breakdown exported from the chloroplast at night. The maltose excess 1 mutant （mex1）, which lacks the chloroplast envelope maltose transporter, accumulates high levels of maltose and starch in chloroplasts and develops a distinctive but previously unexplained chlorotic phenotype as leaves mature. The introduction of additional mutations that prevent starch synthesis, or that block maltose production from starch, also prevent chlorosis of mex1. In contrast, introduction of mutations in disproportionating enzyme （DPE1） results in the accumulation of maltotriose in addition to maltose, and greatly increases chlorosis. These data suggest a link between maltose accumulation and chloroplast homeostasis. Microscopic analyses show that the mesophyll cells in chlorotic mex1 leaves have fewer than half the number of chloroplasts than wild-type cells. Transmission electron microscopy reveals autophagy-like chloroplast degradation in both mex1 and the dpe1/mex1 double mutant. Microarray analyses reveal substantial reprogramming of metabolic and cellular processes, suggesting that organellar protein turnover is increased in mex1, though leaf senescence and senescence-related chlorophyll catabolism are not induced. We propose that the accumulation of maltose and malto-oligosaccharides causes chloroplast dysfunction, which may by signaled via a form of retrograde signaling and trigger chloroplast degradation.