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PubMed2002

Transcriptional effects of the potent enediyne anti-cancer agent Calicheamicin gamma(I)(1).

We have investigated the mode of action of calicheamicin in living cells by using oligonucleotide microarrays to monitor its effects on gene expression across the entire yeast genome. Transcriptional effects were observed as early as 2 min into drug exposure. Among these effects were the upregulation of two nuclear proteins encoding a Y'-helicase (a subtelomerically encoded protein whose function is to maintain telomeres) and a suppressor of rpc10 and rpb40 mutations (both rpc10 and rpb40 encode RNA polymerase subunits). With longer calicheamicin exposure, genes involved in chromatin arrangement, DNA repair and/or oxidative damage, DNA synthesis and cell cycle checkpoint control as well as other nuclear proteins were all differentially expressed. Additionally, ribosomal proteins and a variety of metabolic, biosynthetic, and stress response genes were also altered in their expression.

Aminoglycosides↗
PubMed2001

Cellular responses to DNA damage.

Cells are constantly under threat from the cytotoxic and mutagenic effects of DNA damaging agents. These agents can either be exogenous or formed within cells. Environmental DNA-damaging agents include UV light and ionizing radiation, as well as a variety of chemicals encountered in foodstuffs, or as air- and water-borne agents. Endogenous damaging agents include methylating species and the reactive oxygen species that arise during respiration. Although diverse responses are elicited in cells following DNA damage, this review focuses on three aspects: DNA repair mechanisms, cell cycle checkpoints, and apoptosis. Because the areas of nucleotide excision repair and mismatch repair have been covered extensively in recent reviews, we restrict our coverage of the DNA repair field to base excision repair and DNA double-strand break repair.

Animals↗
PubMed1999

p53 in embryonic development: maintaining a fine balance.

In addition to its role as a tumour suppressor and cell-cycle checkpoint control protein, p53 has been implicated as an important protein in embryonic development. Despite the viability of most p53 null mice, evidence has accumulated that p53 may regulate differentiation and the response of embryonic cells to diverse environmental stresses. Moreover, it appears that maintenance of a fine balance of p53 protein levels within embryonic cells is important for optimal development. Inappropriate overexpression or underexpression of p53 can lead to embryonic lethality or increased risk of malformations. The p53 protein may utilize multiple functional activities in its regulation of developmental processes.

Animals↗
PubMed2005

BRCA1 participates in DNA decatenation.

The tumor suppressor BRCA1 has an important function in the maintenance of genomic stability. Increasing evidence suggests that BRCA1 regulates cell cycle checkpoints and DNA repair after DNA damage. However, little is known about its normal function in the absence of DNA damage. Here we show that BRCA1 interacts and colocalizes with topoisomerase IIalpha in S phase cells. Similar to cells treated with the topoisomerase IIalpha inhibitor ICRF-193, BRCA1-deficient cells show lagging chromosomes, indicating a defect in DNA decatenation and chromosome segregation. More directly, BRCA1 deficiency results in defective DNA decatenation in vitro. Finally, topoisomerase IIalpha is ubiquitinated in a BRCA1-dependent manner, and topoisomerase IIalpha ubiquitination correlates with higher DNA decatenation activity. Together these results suggest an important role of BRCA1 in DNA decatenation and reveal a previously unknown function of BRCA1 in the maintenance of genomic stability.

Antigens, Neoplasm↗
PubMed2006

CDK2-dependent phosphorylation of FOXO1 as an apoptotic response to DNA damage.

The function of cyclin-dependent kinase 2 (CDK2) is often abolished after DNA damage. The inhibition of CDK2 plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences the survival of cells under genotoxic stress is unknown. Forkhead box O (FOXO) transcription factors are emerging as key regulators of cell survival. CDK2 specifically phosphorylated FOXO1 at serine-249 (Ser249) in vitro and in vivo. Phosphorylation of Ser249 resulted in cytoplasmic localization and inhibition of FOXO1. This phosphorylation was abrogated upon DNA damage through the cell cycle checkpoint pathway that is dependent on the protein kinases Chk1 and Chk2. Moreover, silencing of FOXO1 by small interfering RNA diminished DNA damage-induced death in both p53-deficient and p53-proficient cells. This effect was reversed by restored expression of FOXO1 in a manner depending on phosphorylation of Ser249. Functional interaction between CDK2 and FOXO1 provides a mechanism that regulates apoptotic cell death after DNA strand breakage.

Animals↗
PubMed2006

Signaling networks controlled by the MRN complex and MDC1 during early DNA damage responses.

Cells activate complex signaling networks in response to DNA damage. Several proteins and protein complexes are involved in sensing DNA lesions and initiating the DNA damage response networks. The subsequent DNA damage responses, including the initiation of DNA repair pathways, the activation of cell cycle checkpoint controls and the induction of apoptosis, help maintain genomic stability in mammalian systems. Failure to establish the appropriate DNA damage signaling networks results in genomic instability, which is a known causal factor in tumorigenesis. This review will discuss recent progress in the understanding of the mechanisms by which mammalian cells sense DNA lesions and transduce DNA damage signals during early DNA damage responses.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Valproic acid-induced changes in gene expression during neurulation in a mouse model.

The teratogenic potential of valproic acid has been well established both in experimental models and in human clinical studies. As with all human teratogens, there are genetically determined differences in individual susceptibility to the induction of congenital defects. Using a mouse model of valproate-induced neural tube defects, a study was undertaken to examine differential changes in gene expression for selected transcription factor (Pax-3, Emx-1, Emx-2, c-fos, c-jun, creb) and cell cycle checkpoint genes (bcl-2, p53, wee-1) during neural tube closure. In general, exposure to teratogenic concentrations of valproic acid elicited GD 9:12 control levels of transcription factor mRNA expression in GD 9:0 embryos of both strains. This accelerated developmental profile is marked by significant elevation of Emx-1, Emx-2, c-fos, c-jun, and creb expression. There was also a significant over expression of the cell cycle genes p53 and bcl-2 in the LM/Bc embryos in response to the teratogenic insult. Examination of the ratio of expression of these genes clearly favored bcl-2, which supports the hypothesis that altered neuroepithelial cell proliferation rates, rather than increased apoptosis, is the underlying mechanism by which valproic acid alters normal neural tube morphogenesis. An investigation into interactive effects of these genes on the molecular profile of GD 9:0 embryos further validated this observation. That is, the overall proliferative state among the control embryos was prematurely modified into a more differentiated state following teratogenic insult. These results suggest that alterations in the expression of multiple genes are most likely responsible for valproic acid-induced neural tube defects.

Animals↗
PubMed2004

Parathyroid-hormone-related protein as a regulator of pRb and the cell cycle in arterial smooth muscle.

BACKGROUND: Parathyroid hormone-related protein (PTHrP), a normal product of arterial vascular smooth muscle (VSM), contains a nuclear localization signal (NLS) and at least 2 translational initiation sites, one that generates a conventional signal peptide and one that disrupts the signal peptide. These unusual features allow PTHrP either to be secreted in a paracrine/autocrine fashion, and thereby to inhibit arterial smooth muscle proliferation, or to be retained within the cytosol and to translocate into the nucleus, thereby serving as an intracrine stimulator of smooth muscle proliferation. METHODS AND RESULTS: Here, we demonstrate 2 important findings. First, PTHrP dramatically increases the percentage of VSM cells in the S and in G2/M phases of the cell cycle. These effects require critical serine and threonine residues at positions Ser119, Ser130, Thr132, and Ser138 in the carboxy-terminus of PTHrP and are associated with the phosphorylation of the key cell cycle checkpoint regulator retinoblastoma protein, pRb. Second, because PTHrP devoid of the NLS serves as an inhibitor of VSM proliferation, we hypothesized that local delivery of NLS-deleted PTHrP to the arterial wall at the time of angioplasty might prevent neointimal hyperplasia. As hypothesized, using a rat carotid angioplasty model, adenoviral delivery of NLS-deleted PTHrP completely abolished the development of the neointima after angioplasty. CONCLUSIONS: PTHrP interacts with key cell cycle regulatory pathways within the arterial wall. Moreover, NLS-deleted PTHrP delivered to the arterial wall at the time of angioplasty seems to have promise as an agent that could reduce or eliminate the neointimal response to angioplasty.

Adenoviridae↗
PubMed2006

Achaete-scute like 2 (ascl2) is a target of Wnt signalling and is upregulated in intestinal neoplasia.

Achaete-scute like (ASCL)2 is a basic helix-loop-helix transcription factor essential for the maintenance of proliferating trophoblasts during placental development. Using oligonucleotide microarrays we identified ascl2 as a gene significantly upregulated in colorectal adenocarcinomas (n=36 cancers, n=16 normals; 15-fold, P<0.0001). This finding was confirmed by quantitative reverse transcriptase (RT)-PCR on large intestinal cancers (n=29 cancers, n=16 normals; 10-fold, P<0.0001). In situ hybridization for ascl2 demonstrated expression at the base of small and large intestinal crypts (n=304), but in no other normal tissues excepting placenta. By in situ hybridization, 52-71% of colorectal adenomas (n=187), 50-73% of large (n=327) and 33-64% of small intestinal adenocarcinomas (n=124) were positive for ascl2 expression. Upregulation of murine ascl2 was also observed using oligonucleotide microarrays, quantitative RT-PCR and in situ hybridization on apcmin/+ and apc1638N/+ smad4-/+ tumours. Tumour cell lines stably transfected with LEF1(DN) or APC2, or transiently transfected with short-interfering RNA (siRNA) against beta-catenin showed a significant downregulation of ascl2. Colocalization of ascl2 with nuclear beta-catenin was observed in 73 small intestinal adenocarcinomas (P=0.0008) and apcmin/+ tumours. Preliminary in vitro data suggest ascl2 may promote progression through the G2/M cell cycle checkpoint. In summary, ascl2 is a putative regulator of proliferation that is overexpressed in intestinal neoplasia.

Animals↗
PubMed2004

Effects of carbon-ion beams on human pancreatic cancer cell lines that differ in genetic status.

The relative biologic effectiveness (RBE) of carbon-ion beams at 3 different linear energy transfer (LET) values (13, 50, and 80 keV/microm) accelerated by the Heavy Ion Medical Accelerator in Chiba on human pancreatic cancer cell lines differing in genetic status was determined. The RBE values were calculated as D10, the dose (Gy) required to reduce the surviving fraction to 10%, relative to X-rays. We also investigated apoptosis and the relationship between D10 and the cell cycle checkpoint using morphologic examination and flow cytometry analysis, respectively. The RBE values calculated by the D10 values ranged from 1.16 to 1.77 for the 13-keV/microm beam and from 1.83 to 2.46 for the 80-keV/microm beam. A correlation between the D10 values of each cell line and intensity of G2/M arrest was observed. In contrast, LET values did not clearly correlate with induction of apoptosis. These results suggest that carbon-ion beam therapy is a promising modality. Elucidation of the mechanisms of G2/M arrest and apoptosis may provide clues to enhancing the effects of radiation on pancreatic cancer.

Apoptosis↗
PubMed2005

Nibrin functions in Ig class-switch recombination.

Nijmegen breakage syndrome (NBS) is a rare autosomal recessive disorder characterized by predisposition to hematopoietic malignancy, cell-cycle checkpoint defects, and ionizing radiation sensitivity. NBS is caused by a hypomorphic mutation of the NBS1 gene, encoding nibrin, which forms a protein complex with Mre11 and Rad50, both involved in DNA repair. Nibrin localizes to chromosomal sites of class switching, and B cells from NBS patients show an enhanced presence of microhomologies at the sites of switch recombination. Because nibrin is crucial for embryonic survival, direct demonstration by targeted deletion that nibrin functions in class switch recombination has been lacking. Here, we show by cell-type-specific conditional inactivation of Nbn, the murine homologue of NBS1, that nibrin plays a role in the repair of gamma-irradiation damage, maintenance of chromosomal stability, and the recombination of Ig constant region genes in B lymphocytes.

Animals↗
PubMed2005

Truncation of the C-terminus of human MLH1 blocks intracellular stabilization of PMS2 and disrupts DNA mismatch repair.

The human DNA mismatch repair (MMR) protein MLH1 has essential roles in the correction of replication errors and the activation of cell cycle checkpoints and cytotoxic responses to DNA damage that contribute to suppression of cancer risk. MLH1 functions as a heterodimer with the PMS2 protein, and steady state levels of PMS2 are very low in MLH1-deficient cells. Unique to MLH1 among MutL-homolog proteins, and conserved in identified eukaryotic MLH1 proteins, is the so-called C-terminal homology domain (CTH). The function of these C-terminal 20-30 amino acids is not known. We investigated the effect of a C-terminal truncation of human MLH1 (MLH1-L749X) on mammalian MMR by testing its activity in MLH1-deficient cells. We found the CTH to be essential for suppression of spontaneous mutation, activation of a cytotoxic response to 6-thioguanine, and maintenance of normal steady state levels of PMS2. Co-expression in doubly mutant Mlh1-/-; Pms2-/- fibroblasts showed that MLH1-L749X was unable to stabilize PMS2. Over-expression of MLH1-L749X did not reduce stabilization of PMS2 mediated by wild-type MLH1, indicating that truncation of the CTH reduces the ability to compete with wild-type MLH1 for interaction with PMS2. Lack of PMS2 stabilization also was observed with a previously reported pathogenic truncation (MLH1-Y750X), but not with two different point mutations in the CTH. Biochemical assays demonstrated that truncation of the CTH reduced the stability of heterodimers, although MLH1-L749X retained significant capacity for interaction with PMS2. Thus, the CTH of human MLH1 is necessary for error correction, checkpoint signaling, and for promoting interaction with, and the stability of, PMS2. Analysis of the CTH role in stabilizing PMS2 was facilitated by a novel intracellular assay for MLH1-PMS2 interaction. This assay should prove useful for identifying additional amino acids in MLH1 and PMS2 necessary for interaction in cells, and for determining the functional consequences of MLH1 mutations identified in human cancers.

Adaptor Proteins, Signal Transducing↗
PubMed2000

The Drosophila mus101 gene, which links DNA repair, replication and condensation of heterochromatin in mitosis, encodes a protein with seven BRCA1 C-terminus domains.

The mutagen-sensitive-101 (mus101) gene of Drosophila melanogaster was first identified 25 years ago through mutations conferring larval hypersensitivity to DNA-damaging agents. Other alleles of mus101 causing different phenotypes were later isolated: a female sterile allele results in a defect in a tissue-specific form of DNA synthesis (chorion gene amplification) and lethal alleles cause mitotic chromosome instability that can be observed genetically and cytologically. The latter phenotype presents as a striking failure of mitotic chromosomes of larval neuroblasts to undergo condensation of pericentric heterochromatic regions, as we show for a newly described mutant carrying lethal allele mus101(lcd). To gain further insight into the function of the Mus101 protein we have molecularly cloned the gene using a positional cloning strategy. We report here that mus101 encodes a member of the BRCT (BRCA1 C terminus) domain superfamily of proteins implicated in DNA repair and cell cycle checkpoint control. Mus101, which contains seven BRCT domains distributed throughout its length, is most similar to human TopBP1, a protein identified through its in vitro association with DNA topoisomerase IIbeta. Mus101 also shares sequence similarity with the fission yeast Rad4/Cut5 protein required for repair, replication, and checkpoint control, suggesting that the two proteins may be functional homologs.

Amino Acid Sequence↗
PubMed1992

DMC1: a meiosis-specific yeast homolog of E. coli recA required for recombination, synaptonemal complex formation, and cell cycle progression.

DMC1 is a new meiosis-specific yeast gene. Dmc1 protein is structurally similar to bacterial RecA proteins. dmc1 mutants are defective in reciprocal recombination, accumulate double-strand break (DSB) recombination intermediates, fail to form normal synaptonemal complex (SC), and arrest late in meiotic prophase. dmc1 phenotypes are consistent with a functional relationship between Dmc1 and RecA, and thus eukaryotic and prokaryotic mechanisms for homology recognition and strand exchange may be related. dmc1 phenotypes provide further evidence that recombination and SC formation are interrelated processes and are consistent with a requirement for DNA-DNA interactions during SC formation. dmc1 mutations confer prophase arrest. Additional evidence suggests that arrest occurs at a meiosis-specific cell cycle "checkpoint" in response to a primary defect in prophase chromosome metabolism. DMC1 is homologous to yeast's RAD51 gene, supporting the view that mitotic DSB repair has been recruited for use in meiotic chromosome metabolism.

Amino Acid Sequence↗
PubMed1999

Nim1-related kinases coordinate cell cycle progression with the organization of the peripheral cytoskeleton in yeast.

The mechanisms that couple cell cycle progression with the organization of the peripheral cytoskeleton are poorly understood. In Saccharomyces cerevisiae, the Swe1 protein has been shown previously to phosphorylate and inactivate the cyclin-dependent kinase, Cdc28, thereby delaying the onset of mitosis. The nim1-related protein kinase, Hsl1, induces entry into mitosis by negatively regulating Swe1. We have found that Hsl1 physically associates with the septin cytoskeleton in vivo and that Hsl1 kinase activity depends on proper septin function. Genetic analysis indicates that two additional Hsl1-related kinases, Kcc4 and Gin4, act redundantly with Hsl1 to regulate Swe1. Kcc4, like Hsl1 and Gin4, was found to localize to the bud neck in a septin-dependent fashion. Interestingly, hsl1 kcc4 gin4 triple mutants develop a cellular morphology extremely similar to that of septin mutants. Consistent with the idea that Hsl1, Kcc4, and Gin4 link entry into mitosis to proper septin organization, we find that septin mutants incubated at the restrictive temperature trigger a Swe1-dependent mitotic delay that is necessary to maintain cell viability. These results reveal for the first time how cells monitor the organization of their cytoskeleton and demonstrate the existence of a cell cycle checkpoint that responds to defects in the peripheral cytoskeleton. Moreover, Hsl1, Kcc4, and Gin4 have homologs in higher eukaryotes, suggesting that the regulation of Swe1/Wee1 by this class of kinases is highly conserved.

Amino Acid Sequence↗
PubMed1997

Ataxia-telangiectasia and the Nijmegen breakage syndrome: related disorders but genes apart.

Gene mutations provide valuable clues to cellular metabolism. In humans such insights come mainly from genetic disorders. Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are two distinct but closely related, single gene disorders that highlight a complex junction of several signal transduction pathways. These pathways appear to control defense mechanisms against specific types of damage to cellular macromolecules, and probably regulate the processing of certain types of DNA damage or normal intermediates of DNA metabolism. A-T is characterized primarily by cerebellar degeneration, immunodeficiency, genome instability, clinical radiosensitivity, and cancer predisposition. NBS shares all these features except cerebellar deterioration. The cellular phenotypes of A-T and NBS are almost indistinguishable, however, and include chromosomal instability, radiosensitivity, and defects in cell cycle checkpoints normally induced by ionizing radiation. The recent identification of the gene responsible for A-T, ATM, has revealed its product to be a large, constitutively expressed phosphoprotein with a carboxy-terminal region similar to the catalytic domain of phosphatidylinositol 3-kinases (PI 3-kinases). ATM is a member of a family of proteins identified in various organisms, which share the PI 3-kinase domain and are involved in regulation of cell cycle progression and response to genotoxic agents. Some of these proteins, most notably the DNA-dependent protein kinase, have an associated protein kinase activity, and preliminary data indicate this activity in ATM as well. Mutations in A-T patients are null alleles that truncate or destabilize the ATM protein. Atm-deficient mice recapitulate the human phenotype with slower nervous-system degeneration. Two ATM interactors, c-Abl and p53, underscore its role in cellular responses to genotoxic stress. The complexity of ATM's structure and mode of action make it a paradigm of multifaceted signal transduction proteins involved in many physiological pathways via multiple protein-protein interactions. The as yet unknown NBS protein may be a component in an ATM-based complex, with a key role in sensing and processing specific DNA damage or intermediates and signaling their presence to the cell cycle machinery.

Abnormalities, Multiple↗
PubMed2006

Mitogen-activated protein kinase kinase 1-dependent Golgi unlinking occurs in G2 phase and promotes the G2/M cell cycle transition.

Two controversies have emerged regarding the signaling pathways that regulate Golgi disassembly at the G(2)/M cell cycle transition. The first controversy concerns the role of mitogen-activated protein kinase activator mitogen-activated protein kinase kinase (MEK)1, and the second controversy concerns the participation of Golgi structure in a novel cell cycle "checkpoint." A potential simultaneous resolution is suggested by the hypothesis that MEK1 triggers Golgi unlinking in late G(2) to control G(2)/M kinetics. Here, we show that inhibition of MEK1 by RNA interference or by using the MEK1/2-specific inhibitor U0126 delayed the passage of synchronized HeLa cells into M phase. The MEK1 requirement for normal mitotic entry was abrogated if Golgi proteins were dispersed before M phase by treatment of cells with brefeldin A or if GRASP65, which links Golgi stacks into a ribbon network, was depleted. Imaging revealed that unlinking of the Golgi apparatus begins before M phase, is independent of cyclin-dependent kinase 1 activation, and requires MEK signaling. Furthermore, expression of the GRASP family member GRASP55 after alanine substitution of its MEK1-dependent mitotic phosphorylation sites inhibited both late G(2) Golgi unlinking and the G(2)/M transition. Thus, MEK1 plays an in vivo role in Golgi reorganization, which regulates cell cycle progression.

Cell Division↗
PubMed2000

The p53 response to DNA damage in vivo is independent of DNA-dependent protein kinase.

Ionizing radiation (IR) exposure causes mammalian cells to undergo p53-dependent cell cycle arrest and/or apoptosis. The in vivo role of DNA-dependent protein kinase (DNA-PK) in the transduction of the DNA damage signal to p53 remains unresolved. To determine the relationship between DNA-PK and p53, we studied the cell cycle and apoptotic responses to IR in mice deficient in DNA-PK. Using the slip mouse, which harbors an inactivating mutation of the DNA-PK catalytic subunit (DNA-PKcs), we demonstrated not only that these DNA-PKcs null mutants were highly radiosensitive but also that upon IR treatment, p53 accumulated in their cultured cells and tissue. Induced p53 was transcriptionally active and mediated the induction of p21 and Bax in slip cells. Examination of the thymic cell cycle response to IR treatment indicated that the slip G(1)/S-phase cell cycle checkpoint function was intact. We further show that slip mice exhibited a higher level of spontaneous thymic apoptosis as well as a more robust apoptotic response to IR than wild-type mice. Together, these data demonstrate that the p53-mediated response to DNA damage is intact in cells devoid of DNA-PK activity and suggest that other kinases, such as the product of the gene (ATM) mutated in ataxia telangiectasia, are better candidates for regulating IR-induced phosphorylation and accumulation of p53.

Animals↗