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At least 793 records · Page 44Linked to original sources
PubMed2003

Caspase-3-mediated cleavage of Rad9 during apoptosis.

The activation of caspases is a critical event for the execution phase of programmed cell death. Caspases are highly specific in their ability to activate or inhibit many crucial proteins in the cell via cleavage. In this study, we report the identification of several caspase-3-like cleavage sites in the cell-cycle checkpoint protein Rad9. We demonstrate that human Rad9 can be specifically cleaved in cells induced to enter apoptosis by both DNA damage and staurosporine treatment. Indeed, we show that human Rad9 can be effectively cleaved both in vitro and in vivo, which can be inhibited by either a pan-caspase inhibitor or a caspase-3-specific inhibitor. Additionally, no cleavage of Rad9 can be seen in the caspase-3-deficient cell line MCF-7. Site-directed mutagenesis of three of the most conserved cleavage sites dramatically abrogates cleavage of Rad9 by caspase-3 in vitro, and in intact cells after DNA damage. Expression of the cleavage-resistant mutant Rad9 DDD/AAA appears to protect the cell from DNA damage-induced apoptosis. Immunofluorescence studies of Rad9 localization before and after induction of apoptosis show a translocation of Rad9 from the nucleus to the cytosol, concomitant to the appearance of apoptotic morphology. Furthermore, analysis of a truncated Rad9 mutant that corresponds to a putative N-terminal cleavage fragment shows that the N-terminal portion of Rad9 localizes in the cytosol, binds to Bcl-XL, and induces apoptosis. These results support a dual role for cleavage of Rad9: (1) the liberation and translocation of the BH3 domain-containing N-terminus of Rad9 to the cytosol, as a means of promoting apoptosis via antagonism of Bcl-XL, and (2) the disruption of the Rad9-Rad1-Hus1 DNA damage checkpoint complex.

Apoptosis↗
PubMed1999

Mitogen-activated protein kinase kinase 2 activation is essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing radiation.

An increasing body of evidence suggests that mitogen-induced activation of the RAF/ERK signaling pathway is functionally separate from the stress-induced activation of the SEK/JNK/p38 signaling pathway. In general, stress stimuli strongly activate the p38s and the JNKs while only weakly activating ERK1 and ERK2. However, a number of independent groups have now shown that the RAF/ERK signaling pathway is strongly activated by ionizing radiation. In this work, we examine this paradox. We show that both mitogen-activated protein (MAP) kinase kinase 1 (MEK1) and MAP kinase kinase 2 (MEK2) are activated by ionizing radiation. Blockage of this activation through the use of dominant negative MEK2 increases sensitivity of the cell to ionizing radiation and decreases the ability of a cell to recover from the G2/M cell cycle checkpoint arrest. Blocking MEK2 activation does not affect double-strand DNA break repair, however. Although MEK1 is activated to a lesser extent by ionizing radiation, expression of a dominant negative MEK1 does not affect radiation sensitivity of the cell, the G2/M checkpoint of the cell, or double-strand break repair. Because ionizing radiation leads to a different cell cycle arrest (G2/M arrest) than that typically seen with other stress stimuli, and because we have shown that MEK2 can affect G2/M checkpoint kinetics, these results provide an explanation for the observation that the MEKs can be strongly activated by ionizing radiation and only weakly activated by other stressful stimuli.

3T3 Cells↗
PubMed1998

Reduction in the radiation-induced late S phase and G2 blocks in HL-60 cell populations by amiloride, an efficient inhibitor of the Na+/H+ transporter.

Recent investigations that showed that amiloride delayed or inhibited apoptosis indicated it might also attenuate cell cycle checkpoints activated by ionizing radiation. In this report, single- and dual-parameter flow cytometry were used to investigate the effects of amiloride on cell cycle progression, and the effectiveness of amiloride to attenuate the S and G2 phase checkpoint responses induced by 2.5, 5.0, and 7.5 Gy of gamma radiation. The late S-phase delay, noted at 8 h following irradiation, and a radiation-induced G2 block, which was maximum at 16 h after irradiation, were both significantly reduced in amiloride-treated samples. Attenuation of the radiation-induced late S phase and G2 blocks resulted in cell division without apparent apoptosis or necrosis over a 24-h period. Results presented indicate that amiloride reduces the radiation-induced G2 block in HL-60 cell populations almost equally well as caffeine and to a greater extent than staurosporine. Immunofluorescent detection and quantitation of cyclin B1 expression demonstrated that amiloride only significantly reduced cyclin B1 expression following 5.0 Gy, when there was a notable induction of a significant G2 delay, followed by a relatively rapid recovery in cycling potential. The results suggest that amiloride affects the radiation-triggered signaling cascades to alter the kinase activity of proteins associated with mitotic progression, particularly the cyclin B1-p34cdc2 complex. Alternatively, alterations in intracellular ion concentrations induced by amiloride may lead to changes in Ca2+-dependent signaling cascades and thereby decrease the radiation-mediated cell cycle perturbations.

Amiloride↗
PubMed1996

p53 involves cytosine arabinoside-induced apoptosis in cultured cerebellar granule neurons.

The tumor suppressor p53 gene plays a key role in controlling the cell cycle checkpoint and in apoptosis following the exposure of normal cells to DNA damage. To investigate the role of p53 in cytosine arabinoside (Ara C)-induced cell death of CNS neurons, we examined the effect of Ara C on the survival of cultured cerebellar granule neurons from normal wild-type and p53 null mutant mice. When the neurons from wild-type mice were cultured with Ara C, they gradually died after 24 h in culture. In contrast, the neurons from p53 null mutant mice showed a resistance to the Ara C neurotoxicity. These results indicate that p53 involves Ara C-induced apoptosis in cultured cerebellar granule neurons, in which DNA damage may initiate the apoptotic death program of the neurons.

Animals↗
PubMed2003

Overexpression of Krüppel-like factor 4 in the human colon cancer cell line RKO leads to reduced tumorigenecity.

Krüppel-like factor 4 (KLF4) is a zinc-finger-containing transcription factor, the expression of which is enriched in the postmitotic cells of the intestinal epithelium. KLF4 is a target gene of the tumor suppressor adenomatous polyposis coli (APC). We sought to determine the role of KLF4 in suppressing the tumorigenecity of RKO colon cancer cells, which do not express KLF4. We utilized an established system in RKO cells, in which an inducible promoter controls expression of KLF4. Four independent assays were used to assess the effects of KLF4 induction on tumor cells. We find that KLF4 overexpression reduces colony formation, cell migration and invasion, and in vivo tumorigenecity. The mechanism of action of KLF4 does not involve apoptosis. These findings, along with our previous findings that KLF4 induces G1/S arrest, suggest that KLF4 is a cell cycle checkpoint protein that can reduce tumorigenecity of colon cancer cells.

Animals↗
PubMed2003

Ex vivo targeting of p21Cip1/Waf1 permits relative expansion of human hematopoietic stem cells.

Relative quiescence is a defining characteristic of hematopoietic stem cells. Reasoning that inhibitory tone dominates control of stem cell cycling, we previously showed that mice engineered to be deficient in the cyclin-dependent kinase inhibitor, p21Cip1/Waf1 (p21), have an increased stem cell pool under homeostatic conditions. Since p21 was necessary to maintain stem cell quiescence and its absence sufficient to permit increased murine stem cell cycling, we tested whether reduction of p21 alone in human adult-derived stem cells could affect stem cell proliferation. We demonstrate here that interrupting p21 expression ex vivo resulted in expanded stem cell number and in vivo stem cell function compared with control, manipulated cells. Further, we demonstrate full multilineage reconstitution capability in cells where p21 expression was knocked down. Therefore, lifting the brake on cell proliferation by altering cell cycle checkpoints provides an alternative paradigm for increasing hematopoietic stem cell numbers. This approach may be useful for relative ex vivo human stem cell expansion.

ADP-ribosyl Cyclase↗
PubMed1999

Dysregulation of apoptosis in cancer.

Each day, approximately 50 to 70 billion cells perish in the average adult because of programmed cell death (PCD). Cell death in self-renewing tissues, such as the skin, gut, and bone marrow, is necessary to make room for the billions of new cells produced daily. So massive is the flux of cells through our bodies that, in a typical year, each of us will produce and, in parallel, eradicate a mass of cells equal to almost our entire body weight. The morphologic ritual cells go through when experiencing PCD has been termed apoptosis and is executed by a family of intracellular proteases, called caspases. Unlike accidental cell deaths caused by infarction and trauma, these physiologic deaths culminate in fragmentation of cells into membrane-encased bodies which are cleared through phagocytosis by neighboring cells without inciting inflammatory reactions or tissue scarring. Defects in the processes controlling PCD can extend cell life span, contributing to neoplastic cell expansion independently of cell division. Moreover, failures in normal apoptosis pathways contribute to carcinogenesis by creating a permissive environment for genetic instability and accumulation of gene mutations, promoting resistance to immune-based destruction, allowing disobeyance of cell cycle checkpoints that would normally induce apoptosis, facilitating growth factor/hormone-independent cell survival, supporting anchorage-independent survival during metastasis, reducing dependence on oxygen and nutrients, and conferring resistance to cytotoxic anticancer drugs and radiation. Elucidation of the genes that constitute the core machinery of the cell death pathway has provided new insights into tumor biology, revealing novel strategies for combating cancer.

Animals↗
PubMed2002

Regulation of DNA replication fork genes by 17beta-estradiol.

The steroid hormone estrogen can stimulate mitogenesis in hormone-responsive breast cancer epithelial cells. This action is attributed to the transcriptional activity of the ER, a ligand-dependent transcription factor. However, the exact molecular mechanism underlying estrogen-induced proliferation has yet to be completely elucidated. Using custom cDNA microarrays containing many genes implicated in cell cycle progression and DNA replication, we examined the gene expression of a hormone-responsive breast cancer cell line (MCF-7) treated with a mitogenic dose of estrogen in the absence of confounding growth factors found in serum. Gene expression changes were monitored 1, 4, 12, 24, 36, and 48 h after estrogen stimulation so that RNA levels at critical times throughout cell cycle progression could be monitored. Significant changes include the altered transcript levels of genes implicated in transcription, cellular signaling, and cell cycle checkpoints. At time points during which increased numbers of cells were progressing through S phase, a majority of the genes associated with the DNA replication fork were also found to be induced. The coexpression of DNA replication fork genes by estrogen without the support of serum growth factors indicates an important estrogen regulatory component of the molecular mechanism driving estrogen-induced mitogenesis.

Cell Division↗
PubMed2001

Dimethylbenzanthracene carcinogenesis in Gadd45a-null mice is associated with decreased DNA repair and increased mutation frequency.

Mice lacking the Gadd45a gene are susceptible to ionizing radiation-induced tumors. Increased levels of Gadd45a transcript and protein are seen after treatment of cells with ionizing radiation as well as many other agents and treatments that damage DNA. Because cells deficient in Gadd45a were shown to have a partial defect in the global genomic repair component of the nucleotide excision repair pathway of UV-induced photoproducts, dimethylbenzanthracene (DMBA) carcinogenesis was investigated because this agent produces bulky adducts in DNA that are also repaired by nucleotide excision repair. Wild-type mice and mice deficient for Gadd45a were injected with a single i.p. dose of DMBA at 10-14 days of age. The latency for spontaneous deaths was slightly decreased for Gadd45a-null mice compared with wild-type mice. At 17 months, all surviving animals were killed, and similar percentages of each genotype were found to have tumors. However, nearly twice as many Gadd45a-null than wild-type mice had multiple tumors, and three times as many had multiple malignant tumors. The predominant tumor types in wild-type mice were lymphoma and tumors of the intestines and liver. In Gadd45a-null mice, there was a dramatic increase in female ovarian tumors, male hepatocellular tumors, and in vascular tumors in both sexes. In wild-type mice, this dose of DMBA induced a >5-fold increase in Gadd45a transcript in the spleen and ovary, whereas the increase in liver was >20-fold. Nucleotide excision repair, which repairs both UV- and DMBA-induced DNA lesions, was substantially reduced in Gadd45a-null lymphoblasts. Mutation frequency after DMBA treatment was threefold higher in Gadd45a-null liver compared with wild-type liver. Therefore, lack of basal and DMBA-induced Gadd45a may result in enhanced tumorigenesis because of decreased DNA repair and increased mutation frequency. Genomic instability, decreased cell cycle checkpoints, and partial loss of normal growth control in cells from Gadd45a-null mice may also contribute to this process.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1994

Bloom's syndrome cells GM1492 lack detectable p53 protein but exhibit normal G1 cell-cycle arrest after UV irradiation.

The tumor suppressor gene p53 is thought to be a key factor in the onset of G1 cell-cycle arrest following DNA damage. However, here we describe cells derived from a patient with Bloom's syndrome, lacking any detectable p53 protein, that still shows a functional G1 cell-cycle checkpoint after irradiation with UV-C. Comparison with cells from other Bloom's patients showed that the absence of p53 protein is not a specific characteristic of Bloom's syndrome.

Bloom Syndrome↗
PubMed2004

Regulation of Chk2 gene expression in lymphoid malignancies: involvement of epigenetic mechanisms in Hodgkin's lymphoma cell lines.

The tumor suppressor Chk2 kinase plays crucial roles in regulating cell-cycle checkpoints and apoptosis following DNA damage. We investigated the expression levels of the genes encoding Chk2 and several cell-cycle regulators in nine cell lines from lymphoid malignancies, including three Hodgkin's lymphoma (HL) lines. We found that all HL cell lines exhibited a drastic reduction in Chk2 expression without any apparent mutation of the Chk2 gene. However, expression of Chk2 in HL cells was restored following treatment with the histone deacetylase inhibitors trichostatin A (TsA) and sodium butyrate (SB), or with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5Aza-dC). Chromatin-immunoprecipitation (Chip) assays revealed that treatment of HL cells with TsA, SB or 5Aza-dC resulted in increased levels of acetylated histones H3 and H4, and decreased levels of dimethylated H3 lysine 9 at the Chk2 promoter. These results indicate that expression of the Chk2 gene is downregulated in HL cells via epigenetic mechanisms.

Acetylation↗
PubMed1995

Differences in resistance to 5-fluorouracil as a function of cell cycle delay and not apoptosis.

A series of human embryo fibroblasts has previously been shown to display increasing resistance to the antimetabolites methotrexate (MTX) and N-phosphonacetyl-L-aspartate (PALA) with increasing tumorigenicity. This increased resistance was found to be further increased as a result of salvage pathway activity for purine and pyrimidine biosynthesis. A similar pattern of increasing resistance paralleling increasing tumorigenicity has now been shown to occur with 5-fluorouracil (5-FU), which is independent of salvage pathway activity. The KMS normal cell line was found to be more sensitive to 5-FU than either the immortalised KMST or tumorigenic KN-NM cell lines. Immunohistochemical analysis of the three cell lines demonstrated high levels of p53 protein in the KMST and KN-NM cell lines, but undetectable p53 levels in the KMS cell line. From these data it was hypothesised that a difference in p53 function may be causing the difference in the patterns of sensitivity observed in the three cell lines. P53 is now believed to function as a regulator of a G1 to S cell cycle checkpoint and as an inducer of apoptosis following DNA damage to the cell. The differences in sensitivity of the cell lines could not be explained by differences in the levels of apoptosis but could be attributed to differences in cell cycle response. Our evidence suggests that loss of cell cycle control, possibly through loss of p53 function, is an important factor in increasing the drug resistance of fibroblast cell lines.

Antimetabolites, Antineoplastic↗
PubMed1994

Elongation factor 1 delta is enhanced following exposure to ionizing radiation.

We have used the technique of differential display of mRNA to investigate gene expression patterns in a human squamous carcinoma cell line, SCC-35, following exposure to ionizing radiation. We observed that elongation factor 1 delta (EF-1 delta) expression increased within 2 h of radiation exposure with maximum expression at 8 h. These data identify EF-1 delta as a radiation-inducible gene. Cell cycle analysis shows that EF-1 delta expression enhancement precedes G2 arrest in these cells, suggesting that EF-1 delta may participate in the G2-M cell cycle checkpoint.

Base Sequence↗
PubMed2007

Increased common fragile site expression, cell proliferation defects, and apoptosis following conditional inactivation of mouse Hus1 in primary cultured cells.

Targeted disruption of the mouse Hus1 cell cycle checkpoint gene results in embryonic lethality and proliferative arrest in cultured cells. To investigate the essential functions of Hus1, we developed a system for the regulated inactivation of mouse Hus1 in primary fibroblasts. Inactivation of a loxP site-flanked conditional Hus1 allele by using a cre-expressing adenovirus resulted in reduced cell doubling, cell cycle alterations, and increased apoptosis. These phenotypes were associated with a significantly increased frequency of gross chromosomal abnormalities and an S-phase-specific accumulation of phosphorylated histone H2AX, an indicator of double-stranded DNA breaks. To determine whether these chromosomal abnormalities occurred randomly or at specific genomic regions, we assessed the stability of common fragile sites, chromosomal loci that are prone to breakage in cells undergoing replication stress. Hus1 was found to be essential for fragile site stability, because spontaneous chromosomal abnormalities occurred preferentially at common fragile sites upon conditional Hus1 inactivation. Although p53 levels increased after Hus1 loss, deletion of p53 failed to rescue the cell-doubling defect or increased apoptosis in conditional Hus1 knockout cells. In summary, we propose that Hus1 loss leads to chromosomal instability during DNA replication, triggering increased apoptosis and impaired proliferation through p53-independent mechanisms.

Animals↗
PubMed2003

Inactivating mutations targeting the chfr mitotic checkpoint gene in human lung cancer.

A hallmark of cancer is inactivation of cell cycle checkpoints. However, very few mutations targeting mitotic checkpoint genes have been described, and in those instances, a wild-type copy of the gene was retained. chfr is a mitotic checkpoint gene that functions in early prophase delaying chromosome condensation in response to microtubule poisons. In a panel of 53 lung carcinomas for which matched normal tissue was available, we identified three missense mutations in the chfr gene, at least one of which was associated with loss of heterozygosity. In tissue culture checkpoint assays, the tumor-associated missense mutants had reduced activity or were inactive. Together with recent data suggesting that the chfr gene is frequently silenced in various tumors because of methylation of its promoter, these findings suggest that chfr is inactivated by multiple mechanisms in human cancer.

Aged↗
PubMed2003

Apoptosis-like yeast cell death in response to DNA damage and replication defects.

In budding (Saccharomyces cerevisiae) and fission (Schizosaccharomyces pombe) yeast and other unicellular organisms, DNA damage and other stimuli can induce cell death resembling apoptosis in metazoans, including the activation of a recently discovered caspase-like molecule in budding yeast. Induction of apoptotic-like cell death in yeasts requires homologues of cell cycle checkpoint proteins that are often required for apoptosis in metazoan cells. Here, we summarize these findings and our unpublished results which show that an important component of metazoan apoptosis recently detected in budding yeast-reactive oxygen species (ROS)-can also be detected in fission yeast undergoing an apoptotic-like cell death. ROS were detected in fission and budding yeast cells bearing conditional mutations in genes encoding DNA replication initiation proteins and in fission yeast cells with mutations that deregulate cyclin-dependent kinases (CDKs). These mutations may cause DNA damage by permitting entry of cells into S phase with a reduced number of replication forks and/or passage through mitosis with incompletely replicated chromosomes. This may be relevant to the frequent requirement for elevated CDK activity in mammalian apoptosis, and to the recent discovery that the initiation protein Cdc6 is destroyed during apoptosis in mammals and in budding yeast cells exposed to lethal levels of DNA damage. Our data indicate that connections between apoptosis-like cell death and DNA replication or CDK activity are complex. Some apoptosis-like pathways require checkpoint proteins, others are inhibited by them, and others are independent of them. This complexity resembles that of apoptotic pathways in mammalian cells, which are frequently deregulated in cancer. The greater genetic tractability of yeasts should help to delineate these complex pathways and their relationships to cancer and to the effects of apoptosis-inducing drugs that inhibit DNA replication.

Alkylating Agents↗
PubMed1994

Modification of the radiosensitivity of human testicular cancer cells by simian virus 40 sequences.

CRL7800 cells are of human testicular cancer origin and are sensitive to killing by ionizing radiation. After transfection with a plasmid expressing the T-antigen (pSC), cells show enhanced growth and an increased resistance to ionizing radiation. Cell cycle analysis reveals perturbation of a cell cycle checkpoint which, after irradiation, results in an increase in G2-phase arrest in CRL7800VA cells. These experiments demonstrate the modulation of radiation sensitivity and cell cycle arrest of human tumor cells by the introduction of viral genes.

Antigens, Polyomavirus Transforming↗
PubMed2006

Differential effect of selected methylxanthine derivatives on radiosensitization of lung carcinoma cells.

AIM: Using caffeine as a reference derivative, this study was performed to investigate how other methylxanthine derivatives, theophylline, 3-isobutyl-methylxanthine and 1,3-dipropyl-7-methylxanthine, sensitize cells to radiation by modifying cell cycle checkpoints and inducing the apoptotic response. The effect of the methylxanthine derivatives was studied in response to gamma and ultraviolet radiation in a human large cell lung carcinoma cell line, null for p53, a normal lung epithelial cell line and the large cell lung carcinoma cell line stably transfected with p53. METHODS: Effects of theophylline, 3-isobutyl-methylxanthine and 1,3-dipropyl 7-methylxanthine on cell-radiosensitization in comparison to caffeine tested by clonogenic survival assay, MTT assay, ELISA based apoptotic assay, flow cytometry, caspase-3 activity, TUNEL assay, and western blot analysis. RESULTS: All the derivatives, except 3-isobutyl-methylxanthine, increased tumor cell sensitization to radiation by inducing apoptosis in the p53-null lung cancer cell line. The pattern of cell cycle progression revealed that these derivatives increased the number of cells in G1 phase by abrogating the G2/M checkpoint, directing the cells to apoptose through a p53-independent mechanism. In contrast, 3-isobutyl-methylxanthine was more potent than the other derivatives in radiosensitization of normal lung epithelial cells and the lung carcinoma cells stably transfected with wild-type p53. IBMX increased p53 protein level more than caffeine in lung carcinoma cells stably transfected with wild-type p53. CONCLUSION: Our results suggest that 3-isobutyl-methylxanthine might function through a p53-dependent mechanism.

Apoptosis↗