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At least 775 records · Page 43Linked to original sources
PubMed2006

Time-dependent resveratrol-mediated mRNA and protein expression associated with cell cycle in WR-21 cells containing mutated human c-Ha-Ras.

Cancer results from an undesirable imbalance between cellular proliferation and apoptosis. Both processes may be modulated at the level of gene expression, viz., p53 and c-Ha-ras, by dietary bioactive components such as resveratrol. We tested the time-dependent effect of resveratrol on gene and protein expression in WR-21 cells containing a mutated human c-Ha-ras oncogene. We demonstrate cyclic resveratrol-mediated expression of p53, mdm2, p21(cip/waf), Rb, and cyclin G at both the RNA and the protein level at <8 h. However, ras was not differentially expressed at either the RNA or the protein level. p53 was upregulated followed by p21cip/waf, then mdm2, and cyclin G, all downstream p53-activated targets. RNA transcription increased at >8 h for all genes except p53, but protein levels did not suggest uncoupling of transcription and translation. At 24 h, both p53 and Rb expression returned to baseline, suggesting collapse of DNA structure and spindle assembly checkpoints characteristic of mitotic catastrophe. In summary, resveratrol at <8 h induced p53-mediated effects, including apoptosis and cell-cycle arrest (G2/M). However, later, it induced cell-cycle checkpoint dysfunction, indicative of mitotic catastrophe. Thus, future studies should better elucidate the temporal mechanism of the dietary bioactive agent resveratrol on cancer cells.

Animals↗
PubMedDate not supplied

53BP1, an activator of ATM in response to DNA damage.

p53 Binding protein 1 (53BP1) belongs to a family of evolutionarily conserved DNA damage checkpoint proteins with C-terminal BRCT domains and is most likely the human ortholog of the budding yeast Rad9 protein, the first cell cycle checkpoint protein to be described. 53BP1 localizes rapidly to sites of DNA double strand breaks (DSBs) and its initial recruitment to these sites has not been shown to be dependent on any other protein. Initially, 53BP1 was thought to be a mediator of DNA DSB signaling, but now it has been shown to function upstream of ataxia-telangiectasia mutated (ATM), in one of at least two parallel pathways leading to ATM activation in response to DNA damage. Currently, only a single tudor and two BRCT domains are recognized in 53BP1; however, their precise functional role is not understood. Elucidating the function of 53BP1 will be critical to understanding how cells recognize DNA DSBs and how ATM is activated.

Adaptor Proteins, Signal Transducing↗
PubMed2005

PTPRV is a key mediator of p53-induced cell cycle exit.

The p53 tumor suppressor functions as a sequence-specific DNA-binding transcription factor that promotes antiproliferative responses, including cell cycle checkpoints, cellular senescence and apoptosis. The precise nature of the p53 transcriptional programs and the complex mechanisms that govern whether or not a cell dies in response to p53 activation remain elusive. We have recently reported the identification of a new direct p53 target, Ptprv, encoding a transmembrane tyrosine phosphatase. Ptprv expression is dramatically and preferentially increased in cells undergoing p53-dependent cell cycle exit, but not in cells undergoing p53-mediated apoptosis. Importantly, while p53-induced apoptosis is intact in mice lacking Ptprv, Ptprv-null cells are defective in G1 checkpoint control. In addition, we report herein that Ptprv is induced at high cell density and mediates contact inhibition of cell growth. Together, the data suggest that Ptprv is a potent inhibitor of cell proliferation and a critical mediator of p53-induced cell cycle exit.

Animals↗
PubMed2004

Intra-S-phase checkpoint activation by direct CDK2 inhibition.

To ensure proper progression through a cell cycle, checkpoints have evolved to play a surveillance role in maintaining genomic integrity. In this study, we demonstrate that loss of CDK2 activity activates an intra-S-phase checkpoint. CDK2 inhibition triggers a p53-p21 response via ATM- and ATR-dependent p53 phosphorylation at serine 15. Phosphorylation of other ATM and ATR downstream substrates, such as H2AX, NBS1, CHK1, and CHK2 is also increased. We show that during S phase when CDK2 activity is inhibited, there is an unexpected loading of the minichromosome maintenance complex onto chromatin. In addition, there is an increased number of cells with more than 4N DNA content, detected in the absence of p53, suggesting that rereplication can occur as a result of CDK2 disruption. Our findings identify an important role for CDK2 in the maintenance of genomic stability, acting via an ATM- and ATR-dependent pathway.

Animals↗
PubMed1997

The role of inhibitory phosphorylation of CDC2 following DNA replication block and radiation-induced damage in human cells.

It has been suggested that the survival response of p53 defective tumor cells to agents that inhibit DNA replication or damage DNA may be largely dependent on cell cycle checkpoints that regulate the onset of mitosis. In human cells, the mitosis-inducing kinase CDC2/cyclin B is inhibited by phosphorylation of threonine-14 and tyrosine-15, but the roles of these phosphorylations in enforcing checkpoints is not known. We have investigated the situation in a human cervical carcinoma cell line (HeLa cells) and found that low level expression of a mutant nonphosphorylatable form of CDC2 abrogates regulation of the endogenous CDC2/cyclin B. Disruption of this pathway is toxic and renders cells highly sensitive to killing by DNA damage or by inhibition of DNA replication. These findings establish the importance of inhibitory phosphorylation of CDC2 in the survival mechanism used by human cells when exposed to some of the most common forms of anticancer therapy.

CDC2 Protein Kinase↗
PubMed2004

Chk2 drives late G1/early S phase arrest of clonal myeloid progenitors expressing the p210 BCR-ABL tyrosine kinase in response to STI571.

STI571 is the most innovative drug for the cure of Chronic Myeloid Leukemia. It inhibits, in fact, the disease causative event, the p210 bcr-abl tyrosine kinase, and addresses clonal myeloid progenitors to apoptotic death. Here, we demonstrated that STI571 also induces growth arrest by activating the Chk2-Cdc25A-Cdk2 axis, a pathway complementary to p53 in the activation of G(1)/S cell cycle checkpoint. In vitro exposure to STI571 of 32D murine myeloid progenitor cell clones transducing a temperature-sensitive p210 bcr-abl construct was associated with Chk2 phosphorylation and activation, Cdc25A degradation and persistent Cdk2 inhibitory phosphorylation, preventing, in turn, cell transition to and progression throughout the S phase of cell cycle. Chk2 and Cdc25A are both components of a complex network that integrates signals involved in regulated cell cycle progression, DNA repair and cell decision between life or death. Chk2 gene mutations or decreased expression, leading to its protein loss of function on Cdc25A target, and Cdc25A overexpression have been linked to poor prognosis of human cancers. In CML, they might further enhance the proliferative advantage and genomic instability of clonal myeloid progenitors featuring a class of poor prognosis patients eventually resistant to STI571.

Animals↗
PubMed2005

Modulation of cell cycle components by epigenetic and genetic events.

Cell cycle progression is monitored by surveillance mechanisms, or cell cycle checkpoints, that ensure that initiation of a later event is coupled with the completion of an early cell cycle event. Deregulated proliferation is a characteristic feature of tumor cells. Moreover, defects in many of the molecules that regulate the cell cycle have been implicated in cancer formation and progression. Key among these are p53, the retinoblastoma protein (pRb) and its related proteins, p107 and pRb2/p130, and cdk inhibitors (p15, p16, p18, p19, p21, p27), all of which act to keep the cell cycle from progressing until all repairs to damaged DNA have been completed. The pRb (pRb/p16(INK4a)/cyclin D1) and p53 (p14(ARF)/mdm2/p53) pathways are the two main cell-cycle control pathways frequently targeted in tumorigenesis, and the alterations occurring in each pathway depend on the tumor type. Virtually all human tumors deregulate either the pRb or p53 pathway, and oftentimes both pathways simultaneously. This review focuses on the genetic and epigenetic alterations affecting the components of mechanisms regulating the progression of the cell cycle and leading to cancer formation and progression.

Animals↗
PubMed2005

SCFSkp2 complex targeted by Epstein-Barr virus essential nuclear antigen.

The stability of cell cycle checkpoint and regulatory proteins is controlled by the ubiquitin-proteasome degradation machinery. A critical regulator of cell cycle molecules is the E3 ubiquitin ligase SCFSkp2, known to facilitate the polyubiquitination and degradation of p27, E2F, and c-myc. SCFSkp2 is frequently deregulated in human cancers. In this study, we have revealed a novel link between the essential Epstein-Barr virus (EBV) nuclear antigen EBNA3C and the SCFSkp2 complex, providing a mechanism for cell cycle regulation by EBV. EBNA3C associates with cyclin A/cdk2 complexes, disrupting the kinase inhibitor p27 and enhancing kinase activity. The recruitment of SCFSkp2 activity to cyclin A complexes by EBNA3C results in ubiquitination and SCFSkp2-dependent degradation of p27. This is the first report of a viral protein usurping the function of the SCFSkp2 cell cycle regulatory machinery to regulate p27 stability, establishing the foundation for a mechanism by which EBV regulates cyclin/cdk activity in human cancers.

Antigens, Viral↗
PubMed2005

Cell cycle related proteins as prognostic parameters in radically resected non-small cell lung cancer.

BACKGROUND: Experimental evidence suggests that lung cancer development and progression can be linked to an increased proliferation rate. AIMS/METHODS: To evaluate the immunohistochemical expression of seven components of the cell cycle machinery in a series of well characterised non-small cell lung cancer (NSCLC) specimens (n = 105). RESULTS: Multivariate analysis revealed that simultaneous loss of expression of three of these factors--cyclin D1, the cyclin dependent kinase inhibitor p16, and the tumour suppressor retinoblastoma protein Rb2/p130--correlated with survival, confirming the hypothesis that the cyclin D1-p16-retinoblastoma tumour suppressor pathway is inactivated in most lung cancer samples. CONCLUSIONS: These results suggest that loss of control of cell cycle checkpoints is a common occurrence in lung cancer and support the idea that functional cooperation between different cell cycle regulatory proteins constitutes another level of regulation in cell growth control and tumour suppression.

Aged↗
PubMed2005

AMP-activated protein kinase induces a p53-dependent metabolic checkpoint.

Replicative cell division is an energetically demanding process that can be executed only if cells have sufficient metabolic resources to support a doubling of cell mass. Here we show that proliferating mammalian cells have a cell-cycle checkpoint that responds to glucose availability. The glucose-dependent checkpoint occurs at the G(1)/S boundary and is regulated by AMP-activated protein kinase (AMPK). This cell-cycle arrest occurs despite continued amino acid availability and active mTOR. AMPK activation induces phosphorylation of p53 on serine 15, and this phosphorylation is required to initiate AMPK-dependent cell-cycle arrest. AMPK-induced p53 activation promotes cellular survival in response to glucose deprivation, and cells that have undergone a p53-dependent metabolic arrest can rapidly reenter the cell cycle upon glucose restoration. However, persistent activation of AMPK leads to accelerated p53-dependent cellular senescence. Thus, AMPK is a cell-intrinsic regulator of the cell cycle that coordinates cellular proliferation with carbon source availability.

AMP-Activated Protein Kinases↗
PubMed2004

Mechanisms underlying the synergistic effect of SU5416 and cisplatin on cytotoxicity in human ovarian tumor cells.

SU5416 is a selective inhibitor of vascular endothelial growth factor (VEGF) receptors with anti-angiogenesis activity for human cancers. We have previously reported that SU5416 sensitizes ovarian cancer cells to cisplatin via suppression of nucleotide excision repair activity. This study sought to gain further insights into the mechanisms underlying the synergistic effect of SU5416 and cisplatin on cytotoxicity in human ovarian tumor cells. Here, we show that SU5416 inhibited the expression of G1 cell cycle checkpoint regulators, p53, p21, p27 and MDM2 in ovarian carcinoma cells. We also demonstrate that SU5416 triggered the apoptosis of these cells, in addition to augmenting the apoptosis induced by cisplatin, as determined by a Sub-G1 profile analysis using a flow cytometer. Furthermore, we show that SU5416-induced apoptosis is associated with a decrease in the expression of the apoptosis inhibitors, MDM2 and Bcl-2, and an increase in the level of NF-kappaB inhibitor, IkappaBalpha. NF-kappaB is an anti-apoptotic transcription factor, which induces the apoptosis inhibitors, Bcl-XL and IAPs (inhibitor of apoptosis proteins), and IkappaBalpha is an inhibitor of NF-kappaB, which binds to the NF-kappaB and retains it in the cytoplasm. Finally, the compound was found to block cisplatin-induced increases in AP-1 expression and JNK activity, as well as Raf-1 protein level in these cells. Together, these results suggest that the chemosensitizing effect of SU5416 on ovarian tumor cells may be mediated, at least in part, through inhibiting G1 checkpoint control and up-regulating the apoptotic response to cisplatin.

Angiogenesis Inhibitors↗
PubMed2001

Functional impact of concomitant versus alternative defects in the Chk2-p53 tumour suppressor pathway.

Recent evidence identified a genetic and functional link between Chk2 kinase and p53 as a candidate genome integrity checkpoint and a tumour suppressor pathway. Here we report that in human cells, Chk2 and p53 form protein-protein complexes whose abundance increased upon DNA damage, and whose formation was abrogated through cancer associated mutations in the FHA domain of Chk2, or mutations in the tetramerization domain of p53. Whereas among Li-Fraumeni syndrome families mutations of Chk2 or p53 occur in a mutually exclusive manner, we document that the colon cancer cell line HCT-15 concomitantly lacks functions of both Chk2 and p53, the latter demonstrated by a non-invasive reporter assay monitoring p53-dependent transactivation in live cells. Despite the preserved ability of common cancer-derived mutant p53 proteins to bind and potentially 'titrate' activated Chk2, the integrity of the S phase checkpoint response to ionizing radiation remained largely intact and dependent on Chk2 in cells with wild-type, mutant, or no p53. These results provide new mechanistic insights into the Chk2-p53 interplay, suggest how mutations in Chk2 may abrogate its tumour suppressor function, and indicate that compared with individual defects in either Chk2 or p53, concomitant mutations in both of these cell cycle checkpoint regulators may provide some additional selective advantage to tumour cells.

Checkpoint Kinase 2↗
PubMed1995

The checkpoint delaying anaphase in response to chromosome monoorientation is mediated by an inhibitory signal produced by unattached kinetochores.

During mitosis in Ptk1 cells anaphase is not initiated until, on average, 23 +/- 1 min after the last monooriented chromosome acquires a bipolar attachment to the spindle--an event that may require 3 h (Rieder, C. L., A. Schultz, R. W. Cole, and G. Sluder. 1994. J. Cell Biol. 127:1301-1310). To determine the nature of this cell-cycle checkpoint signal, and its site of production, we followed PtK1 cells by video microscopy prior to and after destroying specific chromosomal regions by laser irradiation. The checkpoint was relieved, and cells entered anaphase, 17 +/- 1 min after the centromere (and both of its associated sister kinetochores) was destroyed on the last monooriented chromosome. Thus, the checkpoint mechanism monitors an inhibitor of anaphase produced in the centromere of monooriented chromosomes. Next, in the presence of one monooriented chromosome, we destroyed one kinetochore on a bioriented chromosome to create a second monooriented chromosome lacking an unattached kinetochore. Under this condition anaphase began in the presence of the experimentally created monooriented chromosome 24 +/- 1.5 min after the nonirradiated monooriented chromosome bioriented. This result reveals that the checkpoint signal is not generated by the attached kinetochore of a monooriented chromosome or throughout the centromere volume. Finally, we selectively destroyed the unattached kinetochore on the last monooriented chromosome. Under this condition cells entered anaphase 20 +/- 2.5 min after the operation, without congressing the irradiated chromosome. Correlative light microscopy/elctron microscopy of these cells in anaphase confirmed the absence of a kinetochore on the unattached chromatid. Together, our data reveal that molecules in or near the unattached kinetochore of a monooriented PtK1 chromosome inhibit the metaphase-anaphase transition.

Anaphase↗
PubMed2001

UV-induced skin carcinogenesis in xeroderma pigmentosum group A (XPA) gene-knockout mice with nucleotide excision repair-deficiency.

Nucleotide excision repair (NER) removes a wide variety of lesions from the genome and is deficient in the genetic disorder, xeroderma pigmentosum (XP). In this paper, an in vitro analysis of the XP group A gene product (XPA protein) is reported. Results of an analysis on the pathogenesis of ultraviolet (UV)-B-induced skin cancer in the XPA gene-knockout mouse are also described: (1) contrary to wild type mice, significant bias of p53 mutations to the transcribed strand and no evident p53 mutational hot spots were detected in the skin tumors of XPA-knockout mice. (2) Skin cancer cell lines from UVB-irradiated XPA-knockout mice had a decreased mismatch repair activity and an abnormal cell cycle checkpoint, suggesting that the downregulation of mismatch repair helps cells escape killing by UVB and that mismatch repair-deficient clones are selected for during the tumorigenic transformation of XPA (-/-) cells. (3) The XPA-knockout mice showed a higher frequency of UVB-induced mutation in the rpsL transgene at a low dose of UVB-irradiation than the wild type mice. CC-->TT tandem transition, a hallmark of UV-induced mutation, was detected at higher frequency in the rpsL transgene in the XPA-knockout mice than the wild type mice. This rpsL/XPA mouse system will be useful for further analysing the role of NER in the mutagenesis induced by various carcinogens. (4) The UVB-induced immunosuppression was greatly enhanced in the XPA-knockout mice. It is possible that an enhanced impairment of the immune system by UVB irradiation is involved in the high incidence of skin cancer in XP.

Animals↗
PubMed2001

ATM-dependent dissociation of B55 regulatory subunit from nuclear PP2A in response to ionizing radiation.

Ionizing radiation (IR) is known to activate multiple cell cycle checkpoints that are thought to enhance the ability of cells to respond to DNA damage. Protein phosphatase 2A (PP2A) has been implicated in IR-induced activation of checkpoints; therefore, Jurkat cells were exposed to an activating dose of IR or sham treatment as control, and nuclear extracts were analyzed for PP2A by Mono Q anion exchange chromatography and microcystin affinity chromatography. PP2A exists in eukaryotic cells both as a heterodimer consisting of a 65-kDa scaffolding subunit (A) plus a 36-kDa catalytic subunit (C) and as ABC heterotrimers, containing one of a variety of regulatory (B) subunits. Here we show that IR produces a transient and reversible reduction in the amount of nuclear AB55C heterotrimer without affecting the AB'C heterotrimer or AC heterodimer. In ataxia telangiectasia-mutated (ATM)-deficient cells the amount of nuclear PP2A heterotrimer relative to heterodimer was not reduced by radiation, but the radiation response was restored by transfection of these cells with plasmids encoding ATM. Wortmannin, an inhibitor of kinases such as phosphatidylinositol 3-kinase, also prevented the IR-induced reduction in nuclear PP2A heterotrimer. The changes in nuclear PP2A occurred without a noticeable difference in the carboxyl-terminal methylation of the C subunit, which is known to influence association with B subunits. We conclude a novel ATM-dependent mechanism is regulating association of B55 subunits with nuclear PP2A in response to IR.

Androstadienes↗
PubMed2003

A role of P301L tau mutant in anti-apoptotic gene expression, cell cycle and apoptosis.

In exploring the causative role of the most common Pro(301)-to-Leu (TauP301L) tau missense mutation associated with neurodegenerative tauopathies, we examined TauP301L-mediated apoptotic cell death and the expression of a cluster of genes involved in the inhibition of apoptosis (IAPs) in human neuroblastoma SH-SY5Y cells. Our research found that the expression of TauP301L, but not wild-type tau, down regulated the expression of IAPs, including survivin, which plays a role in the mitotic spindle checkpoint. The inhibition of IAPs coincided with the activation of the pro-apoptotic caspase 3, but preceded apoptotic cell death by TUNEL. Furthermore, TauP301L altered the expression of the cell cycle regulatory proteins and induced the cell cycle arrest at G(2)/M phase. Our studies demonstrate that TauP301L downregulates the expression of genes that protect against apoptosis and regulate cell cycle progression. These results suggest a novel mechanism of apoptotic cell death in TauP301L-expressing cells that involves survivin-mediated activation of cell cycle checkpoint.

Apoptosis↗
PubMed2003

Mitogen-activated protein kinase stimulation of Ca(2+) signaling is required for survival of endoplasmic reticulum stress in yeast.

Endoplasmic reticulum (ER) stress in the budding yeast Saccharomyces cerevisiae triggers Ca2+ influx through a plasma membrane channel composed of Cch1 and Mid1. This response activates calcineurin, which helps to prevent cell death during multiple forms of ER stress, including the response to azole-class antifungal drugs. Herein, we show that ER stress activates the cell integrity mitogen-activate protein kinase cascade in yeast and that the activation of Pkc1 and Mpk1 is necessary for stimulation of the Cch1-Mid1 Ca2+ channel independent of many known targets of Mpk1 (Rlm1, Swi4, Swi6, Mih1, Hsl1, and Swe1). ER stress generated in response to miconazole, tunicamycin, or other inhibitors also triggered a transient G2/M arrest that depended upon the Swe1 protein kinase. Calcineurin played little role in the Swe1-dependent cell cycle arrest and Swe1 had little effect on calcineurin-dependent avoidance of cell death. These findings help to clarify the interactions between Mpk1, calcineurin, and Swe1 and suggest that the calcium cell survival pathway promotes drug resistance independent of both the unfolded protein response and the G2/M cell cycle checkpoint.

Antifungal Agents↗
PubMed2005

CHFR promoter hypermethylation in colon cancer correlates with the microsatellite instability phenotype.

A subset of sporadic colon cancers has been shown to have microsatellite instability caused by an epigenetic inactivation of the MLH1 gene by hypermethylation of the the CpG island in its promoter region. We report here that in colorectal cancer, inactivation of the MLH1 gene is frequently accompanied by hypermethylation of the CpG island in the promoter of the mitotic gene checkpoint with forkhead and ring finger domains (CHFR). This was first observed in the colon cancer cell lines HCT-116, DLD-1, RKO and HT29. Among the 61 primary colon cancer samples studied, hypermethylation of the MLH1 and the CHFR promoter was found in 31% of the tumors. In 68% of all primary cancers (13/19) with MLH1 promoter hypermethylation, hypermethylation of the CHFR promoter was observed as well (P-value < 0.0001, Fisher's two-sided exact). Hypermethylation of the HLTF, MGMT, RASSF1, APC, p14 and p16 promoter regions were also frequent events, being observed in 48% (28/58), 40% (26/64), 21% (14/64), 50% (31/62), 43% (26/60) and 56% (35/63), respectively. However, methylation of these genes was not associated with methylation of either MLH1 or CHFR. The observed methylation profile was unrelated to Duke's stage. The coordinated loss of both mismatch repair caused by methylation of MLH1 and loss of checkpoint control associated with methylation of CHFR suggests the potential to overcome cell cycle checkpoints, which may lead to an accumulation of mutations.

Adaptor Proteins, Signal Transducing↗