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PubMed1998

Molecular mechanisms of DNA double strand break repair.

DNA double-strand breaks (DSBs) are major threats to the genomic integrity of cells. If not taken care of properly, they can cause chromosome fragmentation, loss and translocation, possibly resulting in carcinogenesis. Upon DSB formation, cell-cycle checkpoints are triggered and multiple DSB repair pathways can be activated. Recent research on the Nijmegen breakage syndrome, which predisposes patients to cancer, suggests a direct link between activation of cell-cycle checkpoints and DSB repair. Furthermore, the biochemical activities of proteins involved in the two major DSB repair pathways, homologous recombination and DNA end-joining, are now beginning to emerge. This review discusses these new findings and their implications for the mechanisms of DSB repair.

Animals↗
PubMed2006

Involvement of novel autophosphorylation sites in ATM activation.

ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.

Amino Acid Sequence↗
PubMed2003

The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2.

Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

Animals↗
PubMed1999

Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation.

The Mre11/Rad50 protein complex functions in diverse aspects of the cellular response to double-strand breaks (DSBs), including the detection of DNA damage, the activation of cell cycle checkpoints, and DSB repair. Whereas genetic analyses in Saccharomyces cerevisiae have provided insight regarding DSB repair functions of this highly conserved complex, the implication of the human complex in Nijmegen breakage syndrome reveals its role in cell cycle checkpoint functions. We established mRad50 mutant mice to examine the role of the mammalian Mre11/Rad50 protein complex in the DNA damage response. Early embryonic cells deficient in mRad50 are hypersensitive to ionizing radiation, consistent with a role for this complex in the repair of ionizing radiation-induced DSBs. However, the null mrad50 mutation is lethal in cultured embryonic stem cells and in early developing embryos, indicating that the mammalian Mre11/Rad50 protein complex mediates functions in normally growing cells that are essential for viability.

Animals↗
PubMed2005

Involvement of the p38 MAP kinase in Cr(VI)-induced growth arrest and apoptosis.

Hexavalent chromium [Cr(VI)] is a carcinogenic genotoxin commonly found in industry and the environment. DNA damage resulting from Cr(VI) exposure triggers numerous stress responses, including activation of cell cycle checkpoints and initiation of apoptosis. Mechanisms controlling these responses, while extensively studied, have yet to be fully elucidated. Here, we demonstrate that the p38 mitogen-activated protein kinase (MAPK) is activated by Cr(VI) exposure and that inhibition of p38 function using the selective inhibitor SB203580 results in abrogation of S-phase and G2 cell cycle checkpoints in response to Cr(VI). Also, we observe that inhibition of p38 results in decreased cell survival and increased percentage of apoptotic cells following Cr(VI) treatment. Taken together, these results indicate that p38 function is critical for optimal stress response induced by Cr(VI) exposure.

Apoptosis↗
PubMed1996

Molecular and cellular responses to DNA damage in a murine pituitary adenoma cell line.

Loss of cell cycle control and the inability of the cell to repair DNA at cell cycle checkpoints results in the propagation of genetic lesions which ultimately leads to cancer. To further our understanding of these pathways in pituitary tumorigenesis, we have investigated the effects of DNA damage by gamma radiation in a murine pituitary adenoma (AtT20) cell line with attention to cell cycle checkpoint responses, the induction of apoptosis, and the expression of known regulators of these processes. Irradiated cells exhibited characteristic morphologic changes of apoptosis beginning at 24 h, which included cell shrinkage, chromatin condensation, and cytoplasmic vacuolization, yet the ability to exclude trypan blue was retained for several days. DNA fragmentation could be demonstrated by ethidium bromide staining beginning at 24 h post-irradiation. By propidium iodide staining and flow cytometry, irradiated cells demonstrated G1 and G2 arrest at 24 h, followed at 48 h by a shift to a sub-G1 position of the apoptotic cell population. The G1 arrest coincided with an induction of p53 protein by Western blot analysis which peaked at 4 h post-radiation and persisted beyond 48 h. Expression of c-myc in irradiated cells was found to progressively decrease at 12, 24, and 48 h. Basal expression of the bcl-2 gene in AtT20 cells was found to be 15-fold higher than in normal mouse pituitary by RNase protection assay. Bcl-2 mRNA and protein levels, however, remained unchanged at 24 and 48 h following gamma-irradiation, suggesting that apoptosis occurs independently of bcl-2 gene expression in these cells following this stimulus, as reported in other cell types. We conclude that AtT20 cells undergo G1 and G2 arrest following DNA damage and that a significant proportion of cells then undergo apoptosis. The G1 arrest at 24 h is concurrent with a strong induction of p53 protein, while c-myc expression progressively diminishes. Bcl-2 is highly expressed in this cell line. The absence of variation in bcl-2 expression during apoptosis could be related to its high basal level in these cells.

Adenoma↗
PubMed2001

Perturbation of cell cycle regulators in Peyronie's disease.

Peyronie's disease is a fibromatosis of the tunica albuginea, characterized by development of a plaque consisting primarily of collagen. It has been suggested that trauma to the erect penis is the inciting event. More recent research has focused on the cellular events leading to the dysregulated wound healing and plaque formation. Previous work has shown chromosomal aneusomies and this combined with an increased S-phase in plaque derived cell cultures suggests a perturbation in the cell cycle in this condition. The p53 protein has been shown to be an important cell cycle regulator and pro-apoptotic factor. Aberrant p53 function leading to cell immortalization and proliferation has been implicated in several human malignancies. We hypothesized that abnormal p53 function may explain the high proliferative ability of fibroblasts derived from Peyronie's plaques. This study was undertaken to study the presence and function of p53 and its downstream elements (p21, mdm-2) in Peyronie's disease cell cultures. Plaque-derived fibroblasts have been established in culture and characterized. These cells and control neonatal foreskin fibroblasts were subjected to 5 Gy of gamma radiation to induce DNA damage. After fixation, antibodies to p53 and its transcriptional elements were used to stain irradiated and non-irradiated cells and levels of p53, p21 and mdm-2 were quantified using combined immunofluorescence and flow cytometry. Non-irradiated plaque fibroblasts demonstrated the presence of p53, p21 and mdm-2 at baseline. In control foreskin fibroblasts no p53 or mdm-2 were detectable at baseline. In irradiated foreskin-derived cells significant changes in all elements were demonstrated indicating a fully functional p53 pathway and cell cycle checkpoint system in these cells. In contrast, plaque-derived cells showed no such alterations in levels of cell cycle regulators following irradiation. This is highly suggestive of an aberration of the p53 pathway in plaque-derived fibroblasts. Peyronie's plaque-derived fibroblasts demonstrated stabilization and defunctionalization of p53 protein combined with appropriate responses of its transcriptional elements. These findings may explain the high cell proliferation rates in these cells and suggests a role for perturbation of the p53 pathway in the pathogenesis of Peyronie's disease.

Cell Cycle↗
PubMed2005

Mitochondrial regulation of cell cycle progression during development as revealed by the tenured mutation in Drosophila.

The precise control of the cell cycle requires regulation by many intrinsic and extrinsic factors. Whether the metabolic status of the cell exerts a direct control over cell cycle checkpoints is not well understood. We isolated a mutation, tenured (tend), in a gene encoding cytochrome oxidase subunit Va. This mutation causes a drop in intracellular ATP to levels sufficient to maintain cell survival, growth, and differentiation, but not to enable progression through the cell cycle. Analysis of this gene in vivo and in cell lines shows that a specific pathway involving AMPK and p53 is activated that causes elimination of Cyclin E, resulting in cell cycle arrest. We demonstrate that in multiple tissues the mitochondrion has a direct and specific role in enforcing a G1-S cell cycle checkpoint during periods of energy deprivation.

AMP-Activated Protein Kinases↗
PubMed1998

A human homologue of the Schizosaccharomyces pombe rad1+ checkpoint gene encodes an exonuclease.

In the fission yeast Schizosaccharomyces pombe the rad1(+) gene is required for both the DNA damage-dependent and the DNA replication-dependent cell cycle checkpoints. We have identified a human homologue of the S. pombe rad1(+) gene, designated Hrad1, as well as a mouse homologue: Mrad1. Two Hrad1 alternative splice variants with different open reading frames have been identified; one codes for a long form, Hrad1A, and the other encodes a short form because of N-terminal truncation, Hrad1B. Hrad1A has 60% identity to the S. pombe rad1+ sequence at the DNA level and 49% identity and 72% similarity at the amino acid level. Northern blot analysis indicates elevated levels of expression in testis and cancer cell lines. Chromosomal localization by fluorescence in situ hybridization indicates that Hrad1 is located on chromosome 5p13. 2-13.3. This region is subject to loss of heterozygosity in several human cancers. Hrad1 also shares homology with the Saccharomyces cerevisiae RAD17 and Ustilago maydis REC1 proteins. REC1 has previously been characterized as a 3' --> 5' exonuclease with a C-terminal domain essential for cell cycle checkpoint function. We have expressed and purified polyhistidine-tagged fusions of Hrad1A and Hrad1B and show that HisHrad1A has 3' --> 5' exonuclease activity, whereas HisHrad1B lacks such activity. The biological functions of the two proteins remain to be determined.

Alternative Splicing↗
PubMed1998

The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: linkage of double-strand break repair to the cellular DNA damage response.

Nijmegen breakage syndrome (NBS) is an autosomal recessive disorder characterized by increased cancer incidence, cell cycle checkpoint defects, and ionizing radiation sensitivity. We have isolated the gene encoding p95, a member of the hMre11/hRad50 double-strand break repair complex. The p95 gene mapped to 8q21.3, the region that contains the NBS locus, and p95 was absent from NBS cells established from NBS patients. p95 deficiency in these cells completely abrogates the formation of hMre11/hRad50 ionizing radiation-induced foci. Comparison of the p95 cDNA to the NBS1 cDNA indicated that the p95 gene and NBS1 are identical. The implication of hMre11/hRad50/p95 protein complex in NBS reveals a direct molecular link between DSB repair and cell cycle checkpoint functions.

Acid Anhydride Hydrolases↗
PubMed2003

Loss of XChk1 function triggers apoptosis after the midblastula transition in Xenopus laevis embryos.

Prior to the midblastula transition (MBT), Xenopus laevis embryos do not engage cell cycle checkpoints, although overexpression of the kinase XChk1 arrests cell divisions. At the MBT, XChk1 transiently activates and promotes cell cycle lengthening. In this study, endogenous XChk1 was inhibited by the expression of dominant-negative XChk1 (DN-XChk1). Development appeared normal until the early gastrula stage, when cells lost attachments and chromatin condensed. TUNEL and caspase assays indicated these embryos died by apoptosis during gastrulation. Embryos with unreplicated DNA likewise died by apoptosis. Embryos expressing DN-XChk1 proceeded through additional rapid rounds of DNA replication but initiated zygotic transcription on schedule. Therefore, XChk1 is essential in the early Xenopus embryo for cell cycle remodeling and for survival after the MBT.

Animals↗
PubMed2001

Antisense inhibition of Chk2/hCds1 expression attenuates DNA damage-induced S and G2 checkpoints and enhances apoptotic activity in HEK-293 cells.

The cellular response to DNA damage involves checkpoint controls that delay cell cycle progression in order to provide time for repair of damaged DNA. Chk2/hCds1 is a recently identified homolog of the yeast Cds1 kinase that is involved in cell cycle checkpoint response to DNA damage. To investigate the functions of Chk2/hCds1 in response to DNA damage in mammalian cells, we established a stable human kidney embryonic cell line (HEK-293) that expresses antisense Chk2/hCds1 (Chk2AS) under the control of an inducible promoter. Cells that express Chk2AS display defective S-phase delay in response to DNA replication-mediated DNA damage induced by the topoisomerase I inhibitor camptothecin. The defective G2 checkpoint was also observed in Chk2AS cells exposed to the DNA damaging agent VP-16 or gamma-radiation. Enhanced apoptosis was observed in Chk2AS cells after exposure to gamma-radiation or camptothecin. No p53 activation was observed after DNA damage in HEK-293 or Chk2AS cells. Our results indicate that perturbation of Chk2/hCds1 expression adversely affects the S- and G2-phase checkpoints following DNA damage or DNA replication block, and suggest that reduced expression of Chk2/hCds1 might promote a p53-independent apoptotic response.

Apoptosis↗
PubMed2002

Involvement of the cohesin protein, Smc1, in Atm-dependent and independent responses to DNA damage.

Structural maintenance of chromosomes (SMC) proteins play important roles in sister chromatid cohesion, chromosome condensation, sex-chromosome dosage compensation, and DNA recombination and repair. Protein complexes containing heterodimers of the Smc1 and Smc3 proteins have been implicated specifically in both sister chromatid cohesion and DNA recombination. Here, we show that the protein kinase, Atm, which belongs to a family of phosphatidylinositol 3-kinases that regulate cell cycle checkpoints and DNA recombination and repair, phosphorylates Smc1 protein after ionizing irradiation. Atm phosphorylates Smc1 on serines 957 and 966 in vitro and in vivo, and expression of an Smc1 protein mutated at these phosphorylation sites abrogates the ionizing irradiation-induced S phase cell cycle checkpoint. Optimal phosphorylation of these sites in Smc1 after ionizing irradiation also requires the presence of the Atm substrates Nbs1 and Brca1. These same sites in Smc1 are phosphorylated after treatment with UV irradiation or hydroxyurea in an Atm-independent manner, thus demonstrating that another kinase must be involved in responses to these cellular stresses. Yeast containing hypomorphic mutations in SMC1 and human cells overexpressing Smc1 mutated at both of these phosphorylation sites exhibit decreased survival following ionizing irradiation. These results demonstrate that Smc1 participates in cellular responses to DNA damage and link Smc1 to the Atm signal transduction pathway.

Ataxia Telangiectasia Mutated Proteins↗
PubMed2001

DNA damage invokes mismatch repair-dependent cyclin D1 attenuation and retinoblastoma signaling pathways to inhibit CDK2.

DNA-damage evokes cell cycle checkpoints, which function to maintain genomic integrity. The retinoblastoma tumor suppressor (RB) and mismatch repair complexes are known to contribute to the appropriate cellular response to specific types of DNA damage. However, the signaling pathways through which these proteins impact the cell cycle machinery have not been explicitly determined. RB-deficient murine embryo fibroblasts continued a high degree of DNA replication following the induction of cisplatin damage, but were inhibited for G(2)/M progression. This damage led to RB dephosphorylation/activation and subsequent RB-dependent attenuation of cyclin A and CDK2 activity. In both Rb+/+ and Rb -/- cells, cyclin D1 expression was attenuated following DNA damage. As cyclin D1 is a critical determinant of RB phosphorylation and cell cycle progression, we probed the pathway through which cyclin D1 degradation occurs in response to DNA damage. We found that attenuation of endogenous cyclin D1 is dependent on multiple mismatch repair proteins. We demonstrate that the mismatch repair-dependent attenuation of endogenous cyclin D1 is critical for attenuation of CDK2 activity and induction of cell cycle checkpoints. Together, these studies couple the activity of the retinoblastoma and mismatch repair tumor suppressor pathways through the degradation of cyclin D1 and dual attenuation of CDK2 activity.

Animals↗
PubMed2004

A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to gamma-H2AX foci.

The hereditary disorder ataxia telangiectasia (A-T) is associated with striking cellular radiosensitivity that cannot be attributed to the characterized cell cycle checkpoint defects. By epistasis analysis, we show that ataxia telangiectasia mutated protein (ATM) and Artemis, the protein defective in patients with RS-SCID, function in a common double-strand break (DSB) repair pathway that also requires H2AX, 53BP1, Nbs1, Mre11, and DNA-PK. We show that radiation-induced Artemis hyperphosphorylation is ATM dependent. The DSB repair process requires Artemis nuclease activity and rejoins approximately 10% of radiation-induced DSBs. Our findings are consistent with a model in which ATM is required for Artemis-dependent processing of double-stranded ends with damaged termini. We demonstrate that Artemis is a downstream component of the ATM signaling pathway required uniquely for the DSB repair function but dispensable for ATM-dependent cell cycle checkpoint arrest. The significant radiosensitivity of Artemis-deficient cells demonstrates the importance of this component of DSB repair to survival.

Animals↗
PubMed2000

Nuclear localization and cell cycle-specific expression of CtIP, a protein that associates with the BRCA1 tumor suppressor.

The BRCA1 tumor suppressor has been implicated in a diverse spectrum of cellular processes, including transcriptional regulation, DNA repair, and cell cycle checkpoint control. CtIP was recently identified as a protein that associates with BRCA1 and two other nuclear factors, CtBP1 and Rb1. To understand the functions of CtIP, we have evaluated its biological properties with respect to those of BRCA1. Our results show that CtIP, like its associated factors, is predominantly a nuclear protein. A subset of the endogenous pool of CtIP polypeptides exists in a protein complex that includes both BRCA1 and the BRCA1-associated RING domain protein (BARD1). At the protein level, CtIP expression varies with cell cycle progression in a pattern identical to that of BRCA1. Thus, the steady-state levels of CtIP polypeptides, which remain low in resting cells and G(1) cycling cells, increase dramatically as dividing cells traverse the G(1)/S boundary. In contrast to BRCA1, however, the G(1)/S induction of CtIP expression is mediated primarily by post-transcriptional mechanisms. Finally, the interaction between CtIP and BRCA1 is shown to be stable in the face of genotoxic stress elicited by treatment with UV light, adriamycin, or hydrogen peroxide. Together, these results indicate that CtIP can potentially modulate the functions ascribed to BRCA1 in transcriptional regulation, DNA repair, and/or cell cycle checkpoint control.

Animals↗
PubMed2003

Regulation of cell cycles is of key importance in human papillomavirus (HPV)-associated cervical carcinogenesis.

The rapid progress in molecular biology has allowed the identification of the genes involved in different functions of normal cells and has also improved our understanding of the mechanisms of human carcinogenesis. The human papillomavirus (HPV) is a small double-stranded DNA tumor virus and its genes can manipulate cell cycle control to promote viral persistence and replication. The E6 and E7 proteins of high-risk HPV bind to cell cycle regulatory proteins and interfere with both G1/S and G2/M cell cycle checkpoints much more effectively than the low-risk HPV. The difference between the ability of low and high-risk HPV types to induce immortalization and transformation may well lie in their abilities to interact with the various cell cycle components, resulting in the loss of multiple cell cycle checkpoints, which are important in host genome fidelity, thus potentially resulting in accumulation of genetic abnormalities. Cervical cancer is one of the leading malignancies in women worldwide, with substantial morbidity and mortality. According to current concepts, HPV is recognized as the single most important causal agent in the pathogenesis of this cancer. HPV infection clearly precedes the development of malignancy, while being regularly associated with cervical cancer precursor lesions (all grades of squamous intraepithelial lesions). HPV-infected low-grade squamous intraepithelial lesion (SIL) has three possible outcomes: a) it may regress; b) it can persist; or c) it can make a clinical progression to in situ or invasive carcinoma. It has been well established by prospective cohort studies that the spontaneous regression rate increases in parallel with follow-up duration. In contrast, the clinical progression of lesions usually takes place quite rapidly, i.e. during the first two years from diagnosis. The mechanisms responsible for this divergent clinical behavior of HPV-associated squamous intraepithelial lesions are largely unknown, but currently under intense study in different laboratories worldwide.

Cell Cycle↗
PubMed1999

p21(cip-1/waf-1) deficiency causes deformed nuclear architecture, centriole overduplication, polyploidy, and relaxed microtubule damage checkpoints in human hematopoietic cells.

A recent hypothesis suggests that tumor-specific killing by radiation and chemotherapy agents is due to defects or loss of cell cycle checkpoints. An important component of some checkpoints is p53-dependent induction of p21(cip-1/waf-1). Both p53 and p21 have been shown to be required for microtubule damage checkpoints in mitosis and in G1 phase of the cell cycle and they thus help to maintain genetic stability. We present here evidence that p21(cip-1/waf-1) deficiency relaxes the G1 phase microtubule checkpoint that is activated by microtubule damage induced with nocodazole. Reduced p21(cip-1/waf-1) expression also results in gross nuclear abnormalities and centriole overduplication. p53 has already been implicated in centrosome regulation. Our findings further suggest that the p53/p21 axis is involved in a checkpoint pathway that links the centriole/centrosome cycle and microtubule organization to the DNA replication cycle and thus helps to maintain genomic integrity. The inability to efficiently upregulate p21(cip-1/waf-1) in p21(cip-1/waf-1) antisense-expressing cells in response to microtubule damage could uncouple the centrosome cycle from the DNA cycle and lead to nuclear abnormalicies and polyploidy. A centrosome duplication checkpoint could be a new target for novel chemotherapy strategies.

Cell Cycle↗