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PubMed2004

Analysis of checkpoint responses to histone deacetylase inhibitors.

Cell cycle checkpoints respond to a wide range of stresses to prevent compromise to the integrity of the cell. The best studied checkpoints are those induced by genotoxic agents that cause DNA damage. Histone deacetylase inhibitors not only increase the acetylation state of chromatin histones, but they also perturb the cell cycle, causing both G1 and G2 phase arrests, the latter by initiating a checkpoint response. In this chapter we will describe the analysis of the histone deacetylase inhibitor-sensitive G2 checkpoint using synchronized cell populations.

Bromodeoxyuridine↗
PubMed2002

Vitamin D-mediated growth inhibition of an androgen-ablated LNCaP cell line model of human prostate cancer.

1,25-(OH)(2) vitamin D(3) (1,25-(OH)(2) D), the active metabolite of vitamin D, exerts antiproliferative effects on a variety of tumor cells including prostate. This inhibition requires vitamin D receptors (VDRs) as well as downstream effects on the G1 to S phase checkpoint of the cell cycle. Recent data raise the possibility that androgen plays a role in the antiproliferative effects of 1,25-(OH)(2) D in prostate cancer cells; however, this hypothesis has been difficult to test rigorously as the majority of prostate cancer cell lines (unlike human prostate tumors) lack androgen receptors (ARs). We utilized two different models of androgen-independent prostate cancer that express functional ARs and VDRs to evaluate a possible role of androgen in 1,25-(OH)(2) D mediated growth inhibition. We stably introduced the AR cDNA into the human prostate cancer cell line ALVA 31, which expresses functional VDR but is relatively resistant to growth inhibition by 1,25-(OH)(2) D. Neither ALVA-AR nor the control cells, ALVA-NEO, exhibited substantial growth inhibition by 1,25-(OH)(2) D in the presence or absence of androgen. This observation suggests that the basis for the resistance of ALVA 31 to 1,25-(OH)(2) D-mediated growth inhibition is not the lack of AR. The second model was LNCaP-104R1, an AR-expressing androgen independent prostate cancer cell line derived from androgen dependent LNCaP. 1,25-(OH)(2) D inhibited the growth of LNCaP-104R1 cells in the absence of androgen and this effect was not blocked by the antiandrogen Casodex. As was observed in the parental LNCaP cells, this effect was correlated with G1 phase cell cycle accumulation and upregulation of the cyclin dependent kinase inhibitor (CKI) p27, as well as increased association of p27 with cyclin dependent kinase 2. These findings suggest that the antiproliferative effects of 1,25-(OH)(2) D do not require androgen-activated AR but do involve 1,25-(OH)(2) D induction of CKIs required for G1 cell cycle checkpoint control.

Androgens↗
PubMed2001

Opposite effect of NF-kappa B and c-Jun N-terminal kinase on p53-independent GADD45 induction by arsenite.

Cell cycle checkpoint, a major genomic surveillance mechanism, is an important step in maintaining genomic stability and integrity in response to environmental stresses. Using cells derived from human bronchial epithelial cells, we demonstrate that NF-kappaB and c-Jun N-terminal kinase (JNK) reciprocally regulate arsenic trioxide (arsenite)-induced, p53-independent expression of GADD45 protein, a cell cycle checkpoint protein that arrests cells at the G(2)/M phase transition. Inhibition of NF-kappaB activation by stable expression of a kinase-mutated form of IkappaB kinase caused increased and prolonged induction of GADD45 by arsenite. In contrast, the induction of GADD45 by arsenite was transient and less potent in cells where the NF-kappaB activation pathway was normal. Analysis of the cell cycle profile by flow cytometry indicated that NF-kappaB inhibition potentiates arsenite-induced G(2)/M cell cycle arrest. Abrogation of JNK activation, on the other hand, decreased GADD45 expression induced by arsenite, suggesting a role for JNK activation in GADD45 induction. These results indicate a molecular mechanism by which NF-kappaB and JNK may differentially contribute to cell cycle regulation in response to arsenite.

Arsenites↗
PubMed2004

Analysis of cell cycle regulator proteins in non-small cell lung cancer.

BACKGROUND/AIMS: Abnormalities of the proteins involved in cell cycle checkpoints are extremely common among almost all neoplasms. This study aimed to investigate the expression of four components of the cell cycle machinery-p21, p16, p53, and proliferating cell nuclear antigen (PCNA)-in non-small cell lung cancer (NSCLC). METHODS: The expression of p21, p16, p53, and PCNA was examined in 68 well characterised NSCLC specimens using immunohistochemistry. The coregulation of these proteins and their influence on survival were analysed using both univariate and multivariate analyses. RESULTS: By univariate analysis, the expression of all the proteins examined, except for PCNA, was significantly correlated with survival. In multivariate analysis, the only immunohistochemical parameter able to influence overall survival was p16, confirming the hypothesis that the RB-p16 tumour suppressor pathway is inactivated in most lung cancer samples. Finally, the group of patients with NSCLC who were negative for both p21 and p16 had a significantly shorter overall survival. CONCLUSIONS: These results suggest that loss of control of cell cycle checkpoints is a common occurrence in lung cancers, and support the idea that functional cooperation between different cell cycle inhibitor proteins constitutes another level of regulation in cell growth control and tumour suppression.

Adult↗
PubMed2006

Human Tim/Timeless-interacting protein, Tipin, is required for efficient progression of S phase and DNA replication checkpoint.

Tipin was originally isolated as a protein interacting with Timeless/Tim1/Tim (Tim), which is known to be involved in both circadian rhythm and cell cycle checkpoint regulation. The endogenous Tim and Tipin proteins in human cells, interacting through the N-terminal segment of each molecule, form a complex throughout the cell cycle. Tipin and Tim are expressed in the interphase nuclei mostly at constant levels during the cell cycle, and small fractions are recovered in the chromatin-enriched fractions during S phase. Depletion of endogenous Tipin results in reduced growth rate, and this may be due in part to inefficient progression of S phase and DNA synthesis. Knockdown of Tipin induces radioresistant DNA synthesis and inhibits phosphorylation of Chk1 kinase caused by replication stress, as was observed with that of Tim. Knockdown of Tipin or Tim results in reduced protein level and relocation to the cytoplasm of the respective binding partner, suggesting that the complex formation may be required for stabilization and nuclear accumulation of both proteins. Furthermore, both Tipin and Tim may facilitate the accumulation of Claspin in the nuclei under replication stress, whereas nuclear localization of Tipin and Tim is unaffected by Claspin. Our results indicate that mammalian Tipin is a checkpoint mediator that cooperates with Tim and may regulate the nuclear relocation of Claspin in response to replication checkpoint.

Adaptor Proteins, Signal Transducing↗
PubMed2006

Role of mammalian Rad9 in genomic stability and ionizing radiation response.

Eukaryotic cells have evolved DNA damage response mechanisms utilizing proficient DNA repair and cell cycle checkpoints in order to maintain genomic stability. The Schizosaccharomyces pombe Rad9 gene was initially identified as encoding a cell cycle checkpoint protein. When the mammalian homologue of S. pombe Rad9 was inactivated, however, chromosomal instability was observed even in the absence of DNA damaging agents. Both an increase in chromosome end-to-end associations and telomere loss were observed in cells with inactivated mammalian Rad9. This telomere instability correlated with enhanced S- and G2-phase specific cell killing, delayed kinetics of gamma-H2AX foci appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase specific DNA damage repair. Inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S- and G2-phase of the cell cycle. These newly defined functions of mammalian Rad9 are discussed in relation to telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

Animals↗
PubMedDate not supplied

Radiation-induced phosphorylation of Chk1 at S345 is associated with p53-dependent cell cycle arrest pathways.

Because DNA damage-inducible cell cycle checkpoints are thought to protect cells from the lethal effects of ionizing radiation, a better understanding of the mechanistic functions of cell cycle regulatory proteins may reveal new molecular targets for cancer therapy. The two major regulatory proteins of G2 arrest are Chk1 and p53. Yet, it is unclear how these two proteins interact and coordinate their functional roles during radiation-induced G2 arrest. To determine Chk1's role in p53-dependent G2 arrest, we used p53 proficient cells and examined expression of G2 arrest proteins under conditions in which G2 arrest was inhibited by the staurosporine analog, UCN-01. We found that UCN-01 inhibited both G1 and G2 arrest in irradiated p53 proficient cells. The arrest inhibition was associated with suppression of radiation-induced expression of both p21 and 14-3-3 sigma -- two known p53-dependent G2 arrest proteins. The suppression occurred despite normal induction of p53 and normal phosphorylation of p53 at S20 and Cdc25C at S216 -- the two known substrates of Chk1 kinase activity. In contrast, we showed that radiation-induced phosphorylation of Chk1 at S345 was associated with binding of Chk1 to p53, p21, and 14-3-3 sigma, and that UCN-01 inhibited S345 phosphorylation. We suggest that DNA damage-induced phosphorylation of Chk1 at S345, and subsequent p53 binding, links Chk1 with p53 downstream responses and may provide a coordinated interaction between DNA damage responses and cell cycle arrest functions.

14-3-3 Proteins↗
PubMed1999

Gamma-rays-induced death of human cells carrying mutations of BRCA1 or BRCA2.

There is now evidence to suggest that BRCA1 and BRCA2 are involved in the response of cells to DNA damage and cell cycle checkpoint control. This report examines the death pathways of human cells with various BRCA1 and BRCA2 genotypes after exposure to gamma-rays. A lack of functional BRCA1 and BRCA2 led to defective repair of DNA double-strand breaks in irradiated cells. This impairment resulted in a relaxation of cell cycle checkpoints, production of micronuclei, and a loss of proliferative capacity. Heterozygous BRCA1 and BRCA2 mutations also led to enhanced radiosensitivity, with an impaired proliferative capacity after irradiation. The existence of a phenotype related to radiosensitivity in BRCA1+/- and BRCA2+/- cells raises the question of the response of heterozygous women to radiation.

BRCA1 Protein↗
PubMed2006

Regulation of the p53-MDM2 pathway by 14-3-3 sigma and other proteins.

The 14-3-3 sigma (sigma) protein, a unique member of 14-3-3 family, is a negative regulator of the cell cycle and is induced by p53 to initiate cell cycle checkpoint control after DNA damage. Among the 14-3-3 family members, 14-3-3 sigma is uniquely induced by p53 and has a positive feedback effect on p53 activity in response to DNA damage. Although 14-3-3 sigma is linked to p53-regulated cell cycle checkpoint control, the detailed mechanisms of cell cycle regulation by 14-3-3 sigma remain unclear. Decreased expression of 14-3-3 sigma was reported in several types of carcinomas, suggesting that the negative regulatory role of 14-3-3 sigma in the cell cycle is compromised during tumorigenesis. Given the fact that p53's tumor suppressive function is lost in almost half of all human cancers and that 14-3-3 sigma's activity is linked to the p53 network, a perspective regarding the p53/14-3-3 sigma relationship is needed for cancer research. Here we discuss the mechanisms by which 14-3-3 sigma-stabilizes p53 with the hope that these insights may be applied to develop targeted therapeutic strategies for cancer treatment.

14-3-3 Proteins↗
PubMed2004

Human DNA damage checkpoints and their relevance to soft tissue sarcoma.

Soft tissue sarcoma (STS) is a malignant neoplasm, arising in mesenchymal tissues, that is difficult to treat clinically because it can be highly resistant to chemo-radiotherapy. At present, the mechanism of that resistance remains unclear. Cell cycle checkpoints engender strict control of cell proliferation, arresting the cell cycle to provide time for repair or apoptosis when DNA damage is induced by unprogrammed extrinsic events. These pathways involve at least two checkpoints: one at the G1/S transition and one at the G2/M transition. The p53 gene, which is mutated in several malignant tumors, plays an important role in DNA repair at the G1/S transition; however, there is little information on the G2/M checkpoint in STS. In the present study, several proteins (phospho-p53, -cdc25, -cdc2, -Chk1 and -Chk2) involved in checkpoint pathways were investigated using immunohistochemistry in STS specimens. Most STSs maintain a well-preserved G2/M checkpoint despite the loss of the G1/S checkpoint (phospho-p53: 4.9% (2/41); -cdc25: 41% (17/41); -cdc2: 61% (25/41); -Chk1: 29% (12/41); -Chk2: 46% (19/41)). Furthermore, in a postoperative chemotherapy case the number of cells positive for phospho-cdc25 and -Chk2 was higher in a recurrent tumor than in the primary tumor (n = 7, P = 0.046 < 0.05, Wilcoxon signed-ranks test). These findings indicate that the G2/M checkpoint pathway is well preserved and might contribute to the chemotherapeutic resistance associated with STS.

Adolescent↗
PubMed2000

ATM-dependent phosphorylation and accumulation of endogenous BLM protein in response to ionizing radiation.

Bloom's syndrome (BS), a rare genetic disease, arises through mutations in both alleles of the BLM gene which encodes a 3'-5' DNA helicase identified as a member of the RecQ family. BS patients exhibit a high predisposition to development of all types of cancer affecting the general population and BLM-deficient cells display a strong genetic instability. We recently showed that BLM protein expression is regulated during the cell cycle, accumulating to high levels in S phase, persisting in G2/M and sharply declining in G1, suggesting a possible implication of BLM in a replication (S phase) and/or post-replication (G2 phase) process. Here we show that, in response to ionizing radiation, BLM-deficient cells exhibit a normal p53 response as well as an intact G1/S cell cycle checkpoint, which indicates that ATM and p53 pathways are functional in BS cells. We also show that the BLM defect is associated with a partial escape of cells from the gamma-irradiation-induced G2/M cell cycle checkpoint. Finally, we present data demonstrating that, in response to ionizing radiation, BLM protein is phosphorylated and accumulates through an ATM-dependent pathway. Altogether, our data indicate that BLM participates in the cellular response to ionizing radiation by acting as an ATM kinase downstream effector.

Adenosine Triphosphatases↗
PubMed2006

Spermidine/spermine N1-acetyltransferase overexpression in kidney epithelial cells disrupts polyamine homeostasis, leads to DNA damage, and causes G2 arrest.

Expression of spermidine/spermine N(1)-acetyltransferase (SSAT) increases in kidneys subjected to ischemia-reperfusion injury (IRI). Increased expression of SSAT in vitro leads to alterations in cellular polyamine content, depletion of cofactors and precursors of polyamine synthesis, and reduced cell proliferation. In our model system, a >28-fold increase in SSAT levels in HEK-293 cells leads to depletion of polyamines and elevation in the enzymatic activities of ornithine decarboxylase and S-adenosylmethionine decarboxylase, suggestive of a compensatory reaction to increased polyamine catabolism. Increased expression of SSAT also led to DNA damage and G(2) arrest. The increased DNA damage was primarily due to the depletion of polyamines. Other factors such as increased production of H(2)O(2) due to polyamine oxidase activity may play a secondary role in the induction of DNA lesions. In response to DNA damage the ATM/ATR --> Chk1/2 DNA repair and cell cycle checkpoint pathways were activated, mediating the G(2) arrest in SSAT-expressing cells. In addition, the activation of ERK1 and ERK2, which play integral roles in the G(2)/M transition, is impaired in cells expressing SSAT. These results indicate that the disruption of polyamine homeostasis due to enhanced SSAT activity leads to DNA damage and reduced cell proliferation via activation of DNA repair and cell cycle checkpoint and disruption of Raf --> MEK --> ERK pathways. We propose that in kidneys subjected to IRI, one mechanism through which increased expression of SSAT may cause cellular injury and organ damage is through induction of DNA damage and the disruption of cell cycle.

Acetyltransferases↗
PubMed2006

[Ataxia-telangiectasia: a review].

Ataxia-telangiectasia (AT) is an autosomal recessive inherited disease caused by mutational inactivation of the ATM gene. It is a multisystemic disease, characterized by progressive neurological dysfunction, especially in the cerebellum, oculo-cutaneous telangiectasia, immunodeficiency, recurrent sino-pulmonary infections and high incidence of neoplasms. The responsible gene, ATM, encodes a large protein that belongs to a family of protein kinases with a phosphatidylinositol 3-kinase (Pi3K) domain. ATM is a key regulator of cell cycle checkpoints that causes DNA repair or apoptosis. Several studies report ATM function in target cells (such as neurons, fibroblast, endothelium, germ cells, lymphocytes). The pleiotropic phenotypes of AT reflect the multifaceted activities of ATM protein. In nucleus (lymphocytes, fibroblasts, germ cells) ATM is involved in regulation of cell-cycle checkpoints; in cytoplasm ATM regulates redox state (neurons).

Adolescent↗
PubMed2003

Telomere repeat binding factors: keeping the ends in check.

Per definition, a linear chromosome contains two ends, two sites, which by analogy to double-stranded breaks, might be expected to induce cell cycle checkpoints. The fact that cells divide without inducing such checkpoints suggests that telomeres, the natural ends of linear chromosomes, have the ability to suppress checkpoint activation. This suppression takes place at a number of levels. The TTAGGG repeats of human telomeric DNA recruit telomere specific proteins, among them the telomere repeat binding factors TRF1 and TRF2. These proteins, along with their interaction partners, reorganize the linear chromosome end into a t loop, a protected structure, which hides the very end of the chromosome. Here it is discussed how mammalian telomeres differ from DNA breaks, and what methods they use to prevent checkpoint activation.

Animals↗
PubMed2001

Involvement of Brca1 in S-phase and G(2)-phase checkpoints after ionizing irradiation.

Cell cycle arrests in the G(1), S, and G(2) phases occur in mammalian cells after ionizing irradiation and appear to protect cells from permanent genetic damage and transformation. Though Brca1 clearly participates in cellular responses to ionizing radiation (IR), conflicting conclusions have been drawn about whether Brca1 plays a direct role in cell cycle checkpoints. Normal Nbs1 function is required for the IR-induced S-phase checkpoint, but whether Nbs1 has a definitive role in the G(2)/M checkpoint has not been established. Here we show that Atm and Brca1 are required for both the S-phase and G(2) arrests induced by ionizing irradiation while Nbs1 is required only for the S-phase arrest. We also found that mutation of serine 1423 in Brca1, a target for phosphorylation by Atm, abolished the ability of Brca1 to mediate the G(2)/M checkpoint but did not affect its S-phase function. These results clarify the checkpoint roles for each of these three gene products, demonstrate that control of cell cycle arrests must now be included among the important functions of Brca1 in cellular responses to DNA damage, and suggest that Atm phosphorylation of Brca1 is required for the G(2)/M checkpoint.

BRCA1 Protein↗
PubMed2006

E1B55K-deleted adenovirus (ONYX-015) overrides G1/S and G2/M checkpoints and causes mitotic catastrophe and endoreduplication in p53-proficient normal cells.

In order to take advantage of cell replication machinery, viruses have evolved complex strategies to override cell cycle checkpoints and force host cells into S phase. To do so, virus products must interfere not only with the basal cell cycle regulators, such as pRb or Mad2, but also with the main surveillance pathways such as those controlled by p53 and ATM. Recently, a number of defective viruses has been produced which, lacking the latter ability, are incapable of replicating in normal cells but should be able to grow and finally lyse those cells that, such as the tumor cells, have lost their surveillance mechanisms. A prototype of these oncolytic viruses is the E1B55K-defective Adenovirus ONYX-015, which was predicted to selectively replicate and kill p53-deficient cancer cells. We found that, despite wt p53 and notwithstanding the activation of the checkpoint regulators p53, ATM and Mad2, ONYX-015 actively replicated in HUVEC cells. Furthermore, ONYX-015 replication induced a specific phenotype, which is distinct from that of the E4-deleted adenovirus dlE4 Ad5, although both viruses express the main regulatory region E1A. This phenotype includes overriding of the G(1)/S and G(2)/M checkpoints, over-expression of MAD2 and retardation of mitosis and accumulation of polyploid cells, suggesting the occurrence of alterations at the mitotic-spindle checkpoint and impairment of the post-mitotic checkpoint. Our data suggest that viral E1A and E4 region products can override all host cell-checkpoint response even at the presence of a full activation of the ATM/p53 pathway. Furthermore, the E4 region alone seems to act independently of the E1B55K virus product in impairing the ATM-dependent, p53-independent G(2)/M checkpoint since dlE4 Ad5-infected cells arrested in G(2) while ONYX-015-infected cells did enter mitosis.

Adenoviridae↗
PubMed2002

Alteration of cell cycle in cervical tumor associated with human papillomavirus: cyclin-dependent kinase inhibitors.

The ability of viral oncoproteins to subvert cell cycle checkpoints may constitute a mechanism by which viral oncoproteins induce genetic instability. HPV 16 E6 and E7 disrupt cell cycle checkpoints, particularly affecting nearly all cyclin-dependent kinase inhibitors linked to the G1- and G2- checkpoints, in each case by means of a different mechanism. HPV 16 E7 shows homology with the pRb binding sites of cyclin D1, which consequently releases E2F. In addition, E7 directly binds to p21, and releases PCNA and other S-phase promoting genes. In turn, released E2F activates cyclin E, and cyclin E accelerates p27 proteolysis as a function of the antagonistic reaction of its own inhibitor. The induction of p16 expression is assumed to be indirectly associated with E7, which is upregulated only after prolonged inactivation of Rb. HPV 16 E6 decreased the fidelity of multiple checkpoints controlling both entry into and exit from mitosis, with the mechanism of p53 inactivation. In addition, HPV 16 E6 increased the sensitivity to chemically induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function. Alongside the impressive advances made in the understanding of the molecular mechanisms, which HPV disrupts, the validity of these conclusions should be evaluated in the diagnostic and prognostic fields.

Cyclin-Dependent Kinase Inhibitor p16↗
PubMed2000

The controlling role of ATM in homologous recombinational repair of DNA damage.

The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear. To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells. ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation. Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway. These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation.

Animals↗