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At least 649 records · Page 36Linked to original sources
PubMed1997

Interaction of MAD2 with the carboxyl terminus of the insulin receptor but not with the IGFIR. Evidence for release from the insulin receptor after activation.

We have utilized the yeast two-hybrid system to identify proteins that interact with the cytoplasmic domain of the insulin receptor (IR). We identified a human cDNA encoding a protein that appears to be the human homolog of the yeast MAD2 protein, which we term hMAD2. The yeast MAD2 protein was first identified in a genetic screen to identify cell cycle checkpoint regulatory proteins, yet the mechanism by which MAD2 functions in cell cycle control is currently unclear. Here we show that hMAD2 requires the COOH-terminal 30 amino acids of the IR for interaction and that hMAD2 does not interact with the related insulin-like growth factor I receptor. Interestingly, hMAD2 does not require IR tyrosine autophosphorylation for interaction because it interacts with a kinase-dead IR in the yeast two-hybrid system. In support of this finding, hMAD2-GST fusions were found to interact strongly in vitro with receptors derived from noninsulin-stimulated cells. Furthermore, using two independent in vitro assays, IR activation was found to significantly reduce the interaction of hMAD2 with the IR. Lastly, we show that hMAD2 can be coimmunoprecipitated with the IR from Chinese hamster ovary IR cell lysates, suggesting that this interaction occurs in vivo in cells of mammalian origin. Our results suggest that hMAD2 represents a novel class of proteins that is specific for interaction with the IR as compared with the insulin-like growth factor I receptor and that interacts best with the inactive IR and is released upon receptor autophosphorylation. The function of hMAD2 and its potential role in insulin signaling remain to be elucidated.

Amino Acid Sequence↗
PubMed2000

Cell cycle-coupled variation in topoisomerase IIalpha mRNA is regulated by the 3'-untranslated region. Possible role of redox-sensitive protein binding in mRNA accumulation.

Mammalian topoisomerase IIalpha (Topo II) is a highly regulated enzyme essential for many cellular processes including the G(2) cell cycle checkpoint. Because Topo II gene expression is regulated posttranscriptionally during the cell cycle, we investigated the possible role of the 3'-untranslated region (3'-UTR) in controlling Topo II mRNA accumulation. Reporter assays in stably transfected cells demonstrated that, similar to endogenous Topo II mRNA levels, the mRNA levels of reporter genes containing the Topo II 3'-UTR varied during the cell cycle and were maximal in S and G(2)/M relative to G(1). Topo II 3'-UTR sequence analysis and RNA-protein binding assays identified a 177-nucleotide (base pairs 4772-4949) region containing an AUUUUUA motif sufficient for protein binding. Multiple proteins (84, 70, 44, and 37 kDa) bound this region, and the binding of 84- and 37-kDa (tentatively identified as the adenosine- or uridine-rich element-binding factor AUF1) proteins was enhanced in G(1), correlating with decreased Topo II mRNA levels. The binding activity was enhanced in cellular extracts or cells treated with thiol-reducing agents, and increased binding correlated with decreased Topo II mRNA levels. These results support the hypothesis that cell cycle-coupled Topo II gene expression is regulated by interaction of the 3'-UTR with redox-sensitive protein complexes.

3' Untranslated Regions↗
PubMed2006

Ornithine decarboxylase activity in tumor cell lines correlates with sensitivity to cell death induced by histone deacetylase inhibitors.

Inhibitors of histone deacetylases (HDAC) show significant promise as targeted anticancer agents against a variety of hematologic and solid tumors. HDAC inhibitors arrest the growth of primary cells, but they induce apoptosis or differentiation of tumor cells. Although the precise mechanism is unknown, differences in cell cycle checkpoints and chromatin structure may be responsible. Cellular polyamines regulate both cell cycle progression and chromatin structure. In tumors, polyamines are abundantly produced because of increased activity of the rate-limiting enzyme in polyamine synthesis, ornithine decarboxylase (ODC). To determine if polyamines contribute to the cellular response to HDAC inhibitors, we inhibited ODC activity with alpha-difluoromethylornithine. Polyamine depletion increased resistance to apoptosis induced by HDAC inhibitors. In addition, we found that ODC activity levels correlated with sensitivity to HDAC inhibitors in a panel of tumor cell lines. We conclude that polyamines participate in the cellular response to HDAC inhibitors and that ODC activity correlates with sensitivity to HDAC inhibitor-induced apoptosis. Thus, elevated polyamine levels might be a biomarker for tumor sensitivity to HDAC inhibitor-induced apoptosis. These findings warrant clinical evaluation of tumor samples to determine if high ODC activity levels predict sensitivity to HDAC inhibitors.

Antineoplastic Agents↗
PubMed1998

Relaxed cell-cycle arrests and propagation of unrepaired chromosomal damage in cancer cell lines with wild-type p53.

The role of the p53 protein in mediating G1 and G2 cell-cycle arrests after genotoxic insult has been clearly and reproducibly established in primary diploid fibroblasts, but data obtained from p53 wild-type (wt) cancer cell lines are inconsistent. Furthermore, a large proportion of human tumors have p53 wt genotypes but present genetic aberrations that may result from defective cell-cycle checkpoints. We therefore investigated the integrity of G1/S and G2/M cell-cycle arrests in p53 wt cancer cell lines. In the study presented here, we showed that in most cancer cells tested, G1 arrest was relaxed or absent in comparison with arrest in normal diploid fibroblasts, despite seemingly normal p53 and p21 responses. Two cell lines (MCF7 and HCT116) were synchronized in G0/G1 by leucine starvation and subjected to genotoxic stress to determine more precisely the relative proportion of cells arresting in G1 and G2. Whereas the MCF7 cells showed consistent G1 arrest, the HCT116 cells showed none at all. Furthermore, cell-cycle arrests in G1 and G2 in response to gamma irradiation and bleomycin treatment were transient, as the cells resumed cycling after 48-72 h. The cells resuming proliferation suffered massive apoptosis, but a proportion of the cells were rescued and showed normal doubling times. These cells retained a p53 wt genotype but presented gross chromosomal aberrations in 15-20% of the analyzed metaphases. The aberrations were not clonal. These data show that p53 wt cancer cells have relaxed cell-cycle controls after genotoxic insult and tolerate unrepaired chromosomal damage, despite normal p53 function.

Breast Neoplasms↗
PubMed2005

The Rad50S allele promotes ATM-dependent DNA damage responses and suppresses ATM deficiency: implications for the Mre11 complex as a DNA damage sensor.

Genetic and cytologic data from Saccharomyces cerevisiae and mammals implicate the Mre11 complex, consisting of Mre11, Rad50, and Nbs1, as a sensor of DNA damage, and indicate that the complex influences the activity of ataxia-telangiectasia mutated (ATM) in the DNA damage response. Rad50(S/S) mice exhibit precipitous apoptotic attrition of hematopoietic cells. We generated ATM- and Chk2-deficient Rad50(S/S) mice and found that Rad50(S/S) cellular attrition was strongly ATM and Chk2 dependent. The hypomorphic Mre11(ATLD1) and Nbs1(Delta)(B) alleles conferred similar rescue of Rad50(S/S)-dependent hematopoietic failure. These data indicate that the Mre11 complex activates an ATM-Chk2-dependent apoptotic pathway. We find that apoptosis and cell cycle checkpoint activation are parallel outcomes of the Mre11 complex-ATM pathway. Conversely, the Rad50(S) mutation mitigated several phenotypic features of ATM deficiency. We propose that the Rad50(S) allele is hypermorphic for DNA damage signaling, and that the resulting constitutive low-level activation of the DNA damage response accounts for the partial suppression of ATM deficiency in Rad50(S/S) Atm(-/-) mice.

ATP-Binding Cassette Transporters↗
PubMed2000

p53 checkpoint-defective cells are sensitive to X rays, but not hypoxia.

X-ray-induced damage leads to cell-cycle "checkpoint" arrest by p53-dependent induction of the cyclin-dependent kinase inhibitor p21 (Waf1/Cip1/Sdi1). Human tumor cells that lack this response fail to arrest after exposure to DNA-damaging agents, undergo multiple rounds of endoreduplicative DNA synthesis, and eventually commit to an apoptotic cell death. Since low oxygen tension can also induce p53 protein accumulation, and can lead to cell-cycle arrest or apoptosis, we examined the expression of p21 in tumor cells under normoxic and hypoxic conditions. In a survey of cells, mRNA for the p21 gene was induced two- to threefold in response to hypoxia in a seemingly p53-independent manner. We therefore examined genetically matched cells that differ in their p21 and p53 status for response to ionizing radiation and hypoxia. We found that both p21-deficient and p53-deficient cells exhibit an increase in chromosome instability, an increased level of apoptosis, and a failure to arrest after exposure to ionizing radiation. However, cells that lack either p21 or p53 exhibit no increase in chromosome instability or elevated apoptosis and still arrest in response to hypoxia. Thus, the mechanism responsible for the differential response to either hypoxia or X rays presumably lies in the control of cell-cycle progression in response to stress and its dependence on p21. Since the loss of a DNA-damage-dependent checkpoint does not sensitize cells to killing by stresses that elicit a DNA-damage-independent checkpoint, targeting the function of p21 pharmacologically will not kill tumor cells in situ in the absence of a DNA damage signal.

Apoptosis↗
PubMed2003

DNA damage-induced cell-cycle phase regulation of p53 and p21waf1 in normal and ATM-defective cells.

The ATM-dependent accumulation of p53 and induction of p21waf1 are key events for G1 cell-cycle checkpoint arrest following DNA damage. In ATM-null AT cells, even though the p53 and p21waf1 responses are kinetically delayed and quantitatively reduced, the G1 checkpoint is virtually disrupted, suggesting that these proteins arrive too late in G1 to enforce the arrest. As the precise mechanism remains unclear, we examined the response to DNA double-strand breaks generated by gamma-radiation (IR), to determine if ATM deficiency affects the cell-cycle phase regulation of these molecules. We find that, after irradiation, whereas normal LCL-N cells markedly increase their levels of p53 in all phases of the cell cycle, AT cells fail to show any p53 increase in the G1 phase. In addition, whereas in LCL-N p21waf1 is induced in G1 and G2-M, in AT cells this induction is partly seen in G2-M, but not in G1, indicating a different cell-cycle phase regulation of p53 and p21waf1 as a result of ATM deficiency. The levels and catalytic activity of the p53-targeting kinases ATR and DNA-PK in LCL-N and AT cells are very similar throughout the cell cycle, both before and after IR, thus excluding a phase-specific activity for these kinases. Collectively, our findings demonstrate that, in ATM-deficient cells, the p53-dependent p21waf1 response to DNA damage is not only quantitatively reduced, but also specifically suppressed in the G1 phase, thus providing a mechanistic explanation for the severe disruption of the G1 checkpoint in AT cells.

Ataxia Telangiectasia Mutated Proteins↗
PubMed1999

Human mammary epithelial cells exhibit a differential p53-mediated response following exposure to ionizing radiation or UV light.

The tumor suppressor protein, p53, plays a critical role as a transcriptional activator of downstream target genes involved in the cellular response to DNA damaging agents. We examined the cell cycle checkpoint response of human mammary epithelial cells (HMEC) and their isogenic fibroblast counterparts to ionizing (IR) and ultraviolet (UV) radiation, two genotoxic agents whose DNA damage response pathways involve p53. Using flow cytometric analysis, we found that both mortal and immortalized HMEC, which contain wild-type p53 sequence, do not exhibit a G1 arrest in response to IR, but show an intact G2 checkpoint. Supportive evidence from Western analyses revealed that there was neither an increase in p53 nor one of its downstream targets, p21WAF1, in HMEC exposed to IR. In contrast, isogenic mammary fibroblasts arrest at the G1 checkpoint and induce the p53 and p21WAF1 proteins following IR. By comparison, HMEC exposed to UV displayed an S phase arrest and induced the expression of p53 and p21WAF1. Our results show that the cellular response to DNA damage depends on both the type of damage introduced into the DNA and the specific cell type.

Breast↗
PubMed1999

p53 regulates a G2 checkpoint through cyclin B1.

The p53 tumor suppressor controls multiple cell cycle checkpoints regulating the mammalian response to DNA damage. To identify the mechanism by which p53 regulates G2, we have derived a human ovarian cell that undergoes p53-dependent G2 arrest at 32 degrees C. We have found that p53 prevents G2/M transition by decreasing intracellular levels of cyclin B1 protein and attenuating the activity of the cyclin B1 promoter. Cyclin B1 is the regulatory subunit of the cdc2 kinase and is a protein required for mitotic initiation. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that the cyclin B-dependent G2 checkpoint has a role in preventing neoplastic transformation.

Animals↗
PubMed2004

[The emergent role of p38 MAPKs in the regulation of cell cycle].

The p38 MAPKs are members of the MAPK family. Its pathway has been traditionally associated with the stress and immune response. More recently, it has been reported that the p38 MAPKs pathway can also regulate apoptosis and some differentiation process. It can regulate cellular growth and cell cycle checkpoints in different ways depending on the cell type and the stimulus.

Animals↗
PubMed2000

Drosophila wee1 has an essential role in the nuclear divisions of early embryogenesis.

In Drosophila, the maternally expressed mei-41 and grp genes are required for successful execution of the nuclear division cycles of early embryogenesis. In fission yeast, genes encoding similar kinases (rad3 and chk1, respectively) are components of a cell cycle checkpoint that delays mitosis by inhibitory phosphorylation of Cdk1. We have identified mutations in a gene encoding a Cdk1 inhibitory kinase, Drosophila wee1 (Dwee1). Like mei-41 and grp, Dwee1 is zygotically dispensable but is required maternally for completing the embryonic nuclear cycles. The arrest phenotype of Dwee1 mutants, as well as genetic interactions between Dwee1, grp, and mei-41 mutations, suggest that Dwee1 is functioning in the same regulatory pathway as these genes. These findings imply that inhibitory phosphorylation of Cdk1 by Dwee1 is required for proper regulation of the early syncytial cycles of embryogenesis.

Alleles↗
PubMed2003

Modulation of human checkpoint kinase Chk1 by the regulatory beta-subunit of protein kinase CK2.

Protein kinase CK2 is a serine/threonine protein kinase involved in various aspects of cellular regulation. The regulatory beta-subunit of CK2 exerts a central role not only in mediating formation of tetrameric CK2 complexes but also as a docking partner for several protein kinases. In this study, CK2beta is found to interact with the human cell cycle checkpoint kinase Chk1. The Chk1-interacting region of CK2beta is localized at the C-terminus and the complex between CK2beta and Chk1 is devoid of the catalytic CK2alpha-subunit. The interaction between CK2beta and Chk1 leads to an increase in the Cdc25C phosphorylation activity of Chk1. The screening of several cell lines has revealed that the association between CK2beta and Chk1 also occurs in vivo at a different degree. Collectively, these studies confirm the implication of the regulatory beta-subunit of protein kinase CK2 in cell cycle regulation and identify a novel mechanism for the activation of Chk1 protein kinase.

3T3 Cells↗
PubMed2005

Functional interaction of H2AX, NBS1, and p53 in ATM-dependent DNA damage responses and tumor suppression.

Ataxia-telangiectasia (A-T) mutated (ATM) kinase signals all three cell cycle checkpoints after DNA double-stranded break (DSB) damage. H2AX, NBS1, and p53 are substrates of ATM kinase and are involved in ATM-dependent DNA damage responses. We show here that H2AX is dispensable for the activation of ATM and p53 responses after DNA DSB damage. Therefore, H2AX functions primarily as a downstream mediator of ATM functions in the parallel pathway of p53. NBS1 appears to function both as an activator of ATM and as an adapter to mediate ATM activities after DNA DSB damage. Phosphorylation of ATM and H2AX induced by DNA DSB damage is normal in NBS1 mutant/mutant (NBS1m/m) mice that express an N-terminally truncated NBS1 at lower levels. Therefore, the pleiotropic A-T-related systemic and cellular defects observed in NBS1m/m mice are due to the disruption of the adapter function of NBS1 in mediating ATM activities. While H2AX is required for the irradiation-induced focus formation of NBS1, our findings indicate that NBS1 and H2AX have distinct roles in DNA damage responses. ATM-dependent phosphorylation of p53 and p53 responses are largely normal in NBS1m/m mice after DNA DSB damage, and p53 deficiency greatly facilitates tumorigenesis in NBS1m/m mice. Therefore, NBS1, H2AX, and p53 play synergistic roles in ATM-dependent DNA damage responses and tumor suppression.

Animals↗
PubMed1995

Checkpoints in the cell cycle from a modeler's perspective.

The cell division cycle is a complex process by which cells grow and divide into two viable daughter cells. So that mistakes are not made in this crucial replication process, cells stop at one or more "checkpoints" in the cycle to query their internal state and external conditions, before proceeding to the next stage of the cycle. In this paper we study some simple mathematical models of cell cycle arrest in G1 ("Start") and G2. Our models help to relate the molecular mechanisms of these checkpoints with physiological properties of the cell cycle.

Animals↗
PubMed1998

Explaining differences in sensitivity to killing by ionizing radiation between human lymphoid cell lines.

We surveyed five human hematopoietic cell lines (HSB-2, MOLT-4, Reh, CEM, and HL-60) to determine whether any simple correlates with sensitivity to killing by gamma-irradiation might be revealed. The clonogenic survival gamma-ray dose-response curves for these cell lines cover a wide range of sensitivities. Consistent with previous results for murine hematopoietic cell lines, there was a clear correlation between the rapidity with which irradiation induced apoptosis and clonogenic radiosensitivity of a cell line, although the relationship between timing of apoptosis and radiosensitivity differed between human and murine cell lines. Flow cytometric determination of cell cycle distribution after irradiation showed that differences between human hematopoietic cell lines, in the rate of induction of apoptosis, were generally related to the functioning of cell cycle checkpoints. Whereas the rapidly dying and radiosensitive HSB-2 cell line underwent apoptosis from different points in the cell cycle, the more slowly dying cell lines showed a variety of cell cycle arrest profiles and initiated apoptosis after accumulation of cells in the G2 phase. The lag-phase between arrest in G2 and induction of apoptosis was comparable for MOLT-4, Reh, and CEM; however, HL-60 cells showed a markedly longer G2 arrest that correlated with their greater radioresistance. The results suggest that the total length of time available for DNA damage repair (irrespective of whether this time accrues as blockage in G1, S, or G2), prior to potential activation of apoptosis, is a critical determinant of radiosensitivity in human hematopoietic cell lines. Comparison of the p53 status of these cell lines suggested that mutations in the TP53 gene are contributing to the delay of induction of apoptosis seen in the more radioresistant cell lines. The sensitivity of MOLT-4 and HL-60 cells to killing by DNA-associated 125I decays was determined and was found to correlate with the relative sensitivity of these lines to gamma-irradiation. The highly localized deposition of energy by 125I decays argues that DNA damage is a potent initiator of apoptosis in these cell lines. The results presented suggest that differences in the radiosensitivity of the cell lines examined reflect differences in the rapidity of induction of apoptosis and that radiation-induced cell death in hematopoietic cells can be explained as a response to DNA damage.

Apoptosis↗
PubMed2003

DNA damage during the spindle-assembly checkpoint degrades CDC25A, inhibits cyclin-CDC2 complexes, and reverses cells to interphase.

Cell cycle checkpoints that monitor DNA damage and spindle assembly are essential for the maintenance of genetic integrity, and drugs that target these checkpoints are important chemotherapeutic agents. We have examined how cells respond to DNA damage while the spindle-assembly checkpoint is activated. Single cell electrophoresis and phosphorylation of histone H2AX indicated that several chemotherapeutic agents could induce DNA damage during mitotic block. DNA damage during mitotic block triggered CDC2 inactivation, histone H3 dephosphorylation, and chromosome decondensation. Cells did not progress into G1 but seemed to retract to a G2-like state containing 4N DNA content, with stabilized cyclin A and cyclin B1 binding to Thr14/Tyr15-phosphorylated CDC2. The loss of mitotic cells was not due to cell death because there was no discernible effect on caspase-3 activation, DNA fragmentation, or viability. Extensive DNA damage during mitotic block inactivated cyclin B1-CDC2 and prevented G1 entry when the block was removed. The mitotic DNA damage responses were independent of p53 and pRb, but they were dependent on ATM. CDC25A that accumulated during mitosis was rapidly destroyed after DNA damage in an ATM-dependent manner. Ectopic expression of CDC25A or nonphosphorylatable CDC2 effectively inhibited the dephosphorylation of histone H3 after DNA damage. Hence, although spindle disruption and DNA damage provide conflicting signals to regulate CDC2, the negative regulation by the DNA damage checkpoint could overcome the positive regulation by the spindle-assembly checkpoint.

Animals↗
PubMed1996

Activation of the budding yeast spindle assembly checkpoint without mitotic spindle disruption.

The spindle assembly checkpoint keeps cells with defective spindles from initiating chromosome segregation. The protein kinase Mps1 phosphorylates the yeast protein Mad1p when this checkpoint is activated, and the overexpression of Mps1p induces modification of Mad1p and arrests wild-type yeast cells in mitosis with morphologically normal spindles. Spindle assembly checkpoint mutants overexpressing Mps1p pass through mitosis without delay and can produce viable progeny, which demonstrates that the arrest of wild-type cells results from inappropriate activation of the checkpoint in cells whose spindle is fully functional. Ectopic activation of cell-cycle checkpoints might be used to exploit the differences in checkpoint status between normal and tumor cells and thus improve the selectivity of chemotherapy.

Carrier Proteins↗
PubMed1997

Yeast checkpoint controls and relevance to cancer.

Checkpoint controls arrest cells with defects in DNA replication or DNA damage. For several reasons, checkpoint controls may be relevant to ontogeny and treatment of cancer. Firstly, mutations in two human genes, TP53 and ATM, give rise to cellular defects in cell cycle checkpoints and are associated with cancer. Secondly, although checkpoint defects potentially render the cell damage sensitive, they may do so only in combination with other defects in the cell's response to damage. Therefore, manipulation of checkpoint defects, requiring a description of normal and mutant pathways, will be required for this type of therapeutic approach. Those pathways are being described in yeast cells. In budding yeast, the study of checkpoint genes has led to the view that these genes have many roles in the cellular responses to DNA damage, including roles in arrest in multiple stages of cell cycle, in transcriptional induction of repair genes, in DNA repair itself and additionally some undefined role in DNA replication. The checkpoint pathways and proteins that carry out these responses may consist of sensor proteins that detect damage, signaller proteins that transduce an inhibitory signal and target proteins that are altered to arrest cell division (or cause other changes in cell behaviour). Yeast genes that may act at each step have been identified, leading to a working model of checkpoint pathways. An initial step in the pathway may involve the processing of damage to an intermediate that signals arrest and acts in DNA repair. Human checkpoint pathways may have defects in processing damage as well.

Animals↗