Late G1 Phase Research Articles

Purification and proteomic identification of putative upstream regulators of polo-like kinase-1 from mitotic cell extracts

Polo-like kinase-1 (Plk1) is phosphorylated on Thr210 for activation during mitosis. Here, we investigated the question of which kinase(s) is the specific upstream kinase of mitotic Plk1. Upstream kinases of Plk1 were purified from mitotic cell extracts through column chromatography procedures, and identified by mass spectrometry. Candidates for Plk1 kinase included p21-activated kinase, aurora A, and mammalian Ste20-like kinases. Immunoprecipitates of these proteins from mitotic cell extracts phosphorylated Plk1 on Thr210. Even if the activity of Aurora A was blocked with a specific inhibitor, Plk1 phosphorylation still occurred, suggesting that function of Plk1 could be controlled by these kinases for proper mitotic progression, as well as by Aurora A in very late G2 phase for the beginning of mitosis. Structured abstract MINT- 7996332: PAK1 (uniprotkb: Q13153) physically interacts (MI: 0915) with PLK1 (uniprotkb: P53350) by pull down (MI: 0096) MINT- 7996345: PAK3 (uniprotkb: O75914) physically interacts (MI: 0915) with PLK1 (uniprotkb: P53350) by pull down (MI: 0096)

Involvement of a centrosomal protein kendrin in the maintenance of centrosome cohesion by modulating Nek2A kinase activity

Centrosome cycle is strictly coordinated with chromosome duplication cycle to ensure the faithful segregation of chromosomes. Centrosome duplication occurs from the beginning of S phase, and the duplicated centrosomes are held together by centrosome cohesion to function as a single microtubule organizing center during interphase. At late G2 phase centrosome cohesion is disassembled by Nek2A kinase-mediated phosphorylation and, as a consequence, centrosomes are split and constitute spindle poles in mitosis. It has been reported that depletion of a centrosomal protein kendrin (also named pericentrin) induces premature centrosome splitting in interphase, however, it remains unknown how kendrin contributes to the maintenance of centrosome cohesion. Here we show that kendrin associates with Nek2A kinase, which exhibits considerably low activity. Nek2A kinase activity is inhibited in vitro by addition of the Nek2A-binding region of kendrin in a dose-dependent manner. Furthermore, ectopic expression of the same region decreases the number of the cells with split centrosomes at late G2 phase. Taken together, these results suggest that kendrin anchors Nek2A and suppresses its kinase activity at the centrosomes, and thus, is involved in the mechanism to prevent premature centrosome splitting during interphase.

Genome Instability and Bleomycin Sensitivity Test

Estimation of individual susceptibility to mutagens is often a part of epidemiological studies monitoring the appearance of malignant disease in different populations. Genome exposure to mutagens can lead to DNA damage. The rate of damage depends on individual differences in response, which are usually associated with differences in DNA repair capacity. Cytogenetic studies have shown that the genome of tumour cells is less stable than normal cells and therefore accumulates more damage. Tumour patients show a higher frequency of chromatid and chromosomal aberrations and a predisposition to certain types of tumours. One of the common biomarkers used in monitoring tumour appearance and changed response to DNA damage is the bleomycin test. In its active form, bleomycin (glycopeptid) is a radiomimetic cytostatic that can damage the DNA molecule and cause multiple single and double strands. The bleomycin test is simple and inexpensive, and is based on scoring chromatid breaks in lymphocytes in vitro exposed to bleomycin during the late G2 phase of the cell cycle. This review looks into different factors that may affect test results and discusses its wide implementation in studies of genome instability usually caused by a combination of factors.

Abstract 1073: PIN1 forms a protein complex with Rb2/p130 and controls its phosphorylation status

Abstract Since the majority of somatic cells are quiescent, fine controls of cell cycle re-entry and progression are fundamental for the normal life of the cells. Essential to maintaining the quiescent state is what is termed the “restriction point” (R), the point that divides the early and late G1 phase of cell cycle. The pools of proteins that control this mechanism are still not well-defined. However, one of the major players is the RB gene family (Rb, Rb2/p130 and p107) since ablation of these proteins eliminates R. Because their function, RB proteins are considered tumor suppressor. As common feature, RB proteins share a pocket motif able to bind different E2F transcription factors and then regulating their activity. Differential phosphorylation levels of RB proteins are critical for the binding with their E2F partners and consequently for transcriptional inactivation of target genes during G1 and S phase of cell cycle. Phosphorylation is dependent on cyclin/CDKs complexes: it begins in early-G1 phase where gradual inactivation of RB proteins is required to proceed on S phase of cell cycle. PIN1 belongs to peptidyl-prolyl-isomerase family, which role is to catalyze the cis/trans conformation of Serine/Threonine followed by proline residues and to regulate their activity. PIN1 has been shown to be involved in different signalling pathways, many of which concerning cell cycle progression, cellular proliferation, and neoplastic transformation. In glioblastoma cell lines, Rb2/p130 has been demonstrated to be a major player to control cell cycle progression. Since Rb2/p130 has many Ser-Thr/Pro phospho-sites we hypotesized a possible interaction with PIN1 protein. Co-IP and FAR-western blot experiments demonstrated direct interaction between PIN1 and Rb2/p130. The interaction is phosphorylation dependent, as demonstrated by GST-pulldown experiments on protein lysates treated or not with shrimp alkaline phosphatase. To evaluate the effect of PIN1 on Rb2/p130 protein, scrambled- and Pin1-shRNA were prepared by lentiviral approach. Analysis of asynchronous cells showed not difference in Rb2/p130 protein levels. Since Rb2/p130 is mostly active in G0 phase of cell cycle, cells were synchronized in G0 and G1/S phases. Although the total protein level of Rb2/p130 didn't change, phospho- specific Rb2/p130 antibody showed an increase expression level in asynchronous and G0 phase in PIN1 knockdown cells compare to scramble cells. No protein changes are detected in G1/S phase. As demonstrated in other cellular models, PIN1 may affect the level of CDK/cyclin protein complexes eventually affecting Rb2/p130 phosphorylation. Western blot analysis demonstrated no change in protein level among the major CDK/cyclin proteins that control cell cycle. These results suggest that PIN1 regulates directly the functionality of Rb2/p130 during the G0 and probably the early G1 phase of cell cycle when Rb2/p130 has major activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1073.

Clonally Expanding Thymocytes Having Lineage Capability in Gamma-Ray–Induced Mouse Atrophic Thymus

To characterize, in the setting of gamma-ray-induced atrophic thymus, probable prelymphoma cells showing clonal growth and changes in signaling, including DNA damage checkpoint. A total of 111 and 45 mouse atrophic thymuses at 40 and 80 days, respectively, after gamma-irradiation were analyzed with polymerase chain reaction for D-J rearrangements at the TCRbeta locus, flow cytometry for cell cycle, and Western blotting for the activation of DNA damage checkpoints. Limited D-J rearrangement patterns distinct from normal thymus were detected at high frequencies (43 of 111 for 40-day thymus and 21 of 45 for 80-day thymus). Those clonally expanded thymocytes mostly consisted of CD4(+)CD8(+) double-positive cells, indicating the retention of lineage capability. They exhibited pausing at a late G1 phase of cell cycle progression but did not show the activation of DNA damage checkpoints such as gammaH2AX, Chk1/2, or p53. Of interest is that 17 of the 52 thymuses showing normal D-J rearrangement patterns at 40 days after irradiation showed allelic loss at the Bcl11b tumor suppressor locus, also indicating clonal expansion. The thymocytes of clonal growth detected resemble human chronic myeloid leukemia in possessing self-renewal and lineage capability, and therefore they can be a candidate of the lymphoma-initiating cells.

SBP1 Genetically Interacts With WHI3, a Cell Cycle Control Gene in Saccharyomyces cerevisiae

Proper control of cell cycle is crucial allows cells to grow to the appropriate size in the budding yeast, Saccharyomyces cerevisiae, and to ensure cancer formation does not occur in humans. Although there are a number of cell cycle checkpoints which control passage through the cell cycle, the most critical of these is the G1/S checkpoint, also known as START. Upon passage through the G1/S checkpoint, the cell is committed to replicate its DNA and undergo a round of cell division. In yeast, expression of the cyclin Cln3p, a mammalian cyclin homolog, is necessary to progress through the G1/S checkpoint. Cln3p accumulates to high levels during late G1phase and forms a complex with Cdc28p. The Cln3p/CDC28 complex then enters the nucleus and drives the cell through the G1/S checkpoint.In order to control Cln3p levels through early and middle G1 phase, such that the cell proceeds through the G1/S checkpoint at the appropriate time, it is thought that Whi3p, an RNA recognition motif (RRM) containing protein, translationally represses the CLN3 mRNA. Recently, a genetic interaction was identified between WHI3 and SBP1. SBP1 encodes a single stranded RNA binding protein Sbp1p which has been shown to work in conjunction with Dhh1p repress global mRNA translation under physiological stress. Therefore, it is possible Whi3p may work in conjunction with Sbp1p to translationally repress the CLN3 mRNA during early G1 phase. In this work, we show that overexpression of Sbp1p has a significant effect in whi3Δ cells. First, overexpression of Sbp1p in whi3Δ cells results in a significant growth defect at 37° C. Second, overexpression of Sbp1p in whi3Δ cells results in increased cell size as compared to whi3Δ cells. Next, we will investigate whether Sbp1p is necessary for CLN3 mRNA translational repression.

Molecular mechanisms underlying nucleocytoplasmic shuttling of actinin-4

In addition to its well-known role as a crosslinker of actin filaments at focal-adhesion sites, actinin-4 is known to be localized to the nucleus. In this study, we reveal the molecular mechanism underlying nuclear localization of actinin-4 and its novel interactions with transcriptional regulators. We found that actinin-4 is imported into the nucleus through the nuclear pore complex in an importin-independent manner and is exported by the chromosome region maintenance-1 (CRM1)-dependent pathway. Nuclear actinin-4 levels were significantly increased in the late G2 phase of the cell cycle and were decreased in the G1 phase, suggesting that active release from the actin cytoskeleton was responsible for increased nuclear actinin-4 in late G2. Nuclear actinin-4 was found to interact with the INO80 chromatin-remodeling complex. It also directs the expression of a subset of cell-cycle-related genes and interacts with the upstream-binding factor (UBF)-dependent rRNA transcriptional machinery in the M phase. These findings provide molecular mechanisms for both nucleocytoplasmic shuttling of proteins that do not contain a nuclear-localization signal and cell-cycle-dependent gene regulation that reflects morphological changes in the cytoskeleton.

Radioprotection: the non-steroidal anti-inflammatory drugs (NSAIDs) and prostaglandins.

Clinical and experimental studies of the acute and late effects of radiation on cells have enhanced our knowledge of radiotherapy and have led to the optimisation of radiation treatment schedules and to more precise modes of radiation delivery. However, as both normal and cancerous tissues have similar response to radiation exposure, radiation-induced injury on normal tissues may present either during, or after the completion of, the radiotherapy treatment. Studies on both NSAIDs and prostaglandins have indeed shown some evidence of radioprotection. Both have the potential to increase the survival of cells but by entirely different mechanisms. Studies of cell kinetics reveal that cells in the mitotic (M) and late G2 phases of the cell cycle are generally most sensitive to radiation compared with cells in the early S and G1/G0 phases. Furthermore, radiation leads to a mitotic delay in the cell cycle. Thus, chemical agents that either limit the proportion of cells in the M and G2 phases of the cell cycle or enhance rapid cell growth could in principle be exploited for their potential use as radioprotectors to normal tissue during irradiation. NSAIDs have been shown to exert anti-cancer effects by causing cell-cycle arrest, shifting cells towards a quiescence state (G0/G1). The same mechanism of action was observed in radioprotection of normal tissues. An increase in arachidonic acid concentrations after exposure to NSAIDs also leads to the production of an apoptosis-inducer ceramide. NSAIDs also elevate the level of superoxide dismutase in cells. Activation of heat shock proteins by NSAIDs increases cell survival by alteration of cytokine expression. A role for NSAIDs with respect to inhibition of cellular proliferation possibly by an anti-angiogenesis mechanism has also been suggested. Several in-vivo studies have provided evidence suggesting that NSAIDs may protect normal tissues from radiation injury. Prostaglandins do not regulate the cell cycle, but they do have a variety of effects on cell growth and differentiation. PGE(2) mediates angiogenesis, increasing the supply of oxygen and nutrients, essential for cellular survival and growth. Accordingly, PGE(2) at sufficiently high plasma concentrations enhances cellular survival by inhibiting pro-inflammatory cytokines such as TNF-alpha and IL-1beta. Thus, PGE(2) acts as a modulator, rather than a mediator, of inflammation. Prospective studies have suggested the potential use of misoprostol, a PGE(1) analogue, before irradiation, in prevention of radiation-induced side effects. The current understanding of the pharmacology of NSAIDs and prostaglandins shows great potential to minimise the adverse effects of radiotherapy on normal tissue.

Liquid chromatography/mass spectrometry analysis revealing preferential occurrence of non‐arachidonate‐containing phosphatidylinositol bisphosphate species in nuclei and changes in their levels during cell cycle

Phosphatidylinositol phosphates (PtdInsPs) are present within the nucleus, as well as in the membrane. In this mass spectrometry study, different acyl-containing species of endonuclear PtdInsPs were analyzed in order to clearly understand the role of individual molecular species. A (34:1) acyl-containing phosphatidylinositol bisphosphate [PtdInsP(2)(34:1)] and PtdInsP(2)(36:1) were preferentially detected in envelope-less nuclei prepared from various cultured human cells, while PtdInsP(2)(38:4) was not a major component within these nuclei. A significant amount of PtdInsP(2)(34:0) was detected in the HeLa cell nucleus, but not in the A431 and THP-1 cell nuclei. During the cell cycle in HeLa cells, PtdInsP(2)(34:0) levels increased in the early G1 phase, and then gradually decreased through S phase, while PtdInsP(2)(34:1) levels tended to decrease only in late G1 phase and PtdInsP(2)(38:4) did not change significantly. Thus, individual PtdInsP(2) species apparently play different roles in nuclear events based on individual regulation of endonuclear levels. The non-arachidonate-containing species were also detected in normal human blood and fluids, suggesting that these minor species may have unique functions in the human body. The techniques used in this study will be applied to clinical studies on a PtdInsPs metabolism.

Function of HES6 an Inhibitor of HES1, in the Breast Cancer Cell Lines T47D and MCF-7 Is to Up-Regulate E2F-1 and Increased Proliferation.

Abstract Hormone resistance remains as a major obstacle in breast cancer treatment. The majority of breast tumors are dependent on estrogens to grow, therefore anti-estrogens such as Tamoxifen is an effective treatment option. We have found that a transcription factor Hes6, earlier described in the nervous tissue, is up-regulated in hormone-independent breast cancer cells compared to normal estrogen sensitive breast cancer cells. By using a lenti-virus system, we have over-expressed Hes6 in T47D cells which are estrogen sensitive. This resulted in increased proliferation. We also found that estrogen treatment of MCF-7 breast cancer cells induced expression of endogenous Hes6 in the G1-phase. We could not find any binding of estrogen receptor α to the HES6 gene but instead found a binding site up-steam of the promoter in the ASCL1 gene. ASCL1 is a known inducer of HES6 and we are showing that ASCL1 is up-regulated by 17β-estradiol in MCF-7 cells. To find the mechanism behind the tumorogenic effects of Hes6, we analysed important factors of the cell-cycle. We found that the G1-phase factor E2F-1 was up-regulated in response to increased Hes6 expression. Since HES6 have been shown to be an inhibitor of HES1, this is in agreement with a previous study where we found that HES1 inhibited proliferation by binding to the promoter of and down-regulating E2F-1 expression. E2F-1 is an important limiting factor in late G1-phase of the cell cycle and can drive cell proliferation.We believe that the HES1-HES6 interplay is important in antiestrogen-resistant breast cancer. Studies are now ongoing to investigate if Hes6 is up-regulated in estrogen-independent human breast cancer samples. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2153.

NIPA Phosphorylation and Inactivation at G2/M Is Mediated by ERK2.

Abstract 2513Poster Board II-490Regulated oscillation of protein expression is an essential mechanism of cell cycle control. The SCF class of E3 ubiquitin ligases is involved in this process by targeting cell cycle regulatory proteins for degradation by the proteasome. We previously reported cloning of NIPA (Nuclear Interaction Partner of ALK) in complex with constitutively active oncogenic fusions of ALK, contributing to the development of lymphomas and sarcomas. Subsequently we characterized NIPA as a F-Box protein that defines an oscillating ubiquitin E3-ligase. The SCF-NIPA complex targets nuclear cyclin B1 for ubiquitination in interphase while phosphorylation of NIPA in late G2 phase and mitosis inactivates the complex to allow for accumulation of cyclin B1, a critical event for proper G2/M transition. Thus, SCF-NIPA executes an important G2/M checkpoint control. We recently specified three serine residues Ser 354, 359 and 395 implicated in NIPA phosphorylation at G2/M. These data suggest a sequential NIPA phosphorylation, where initial Ser 354 and 359 phosphorylation is most crucial for SCF-NIPA inactivation by dissociating the SCF-NIPA complex. Here we aimed to find the kinase responsible in this initial most important phosphorylation step. Using in vitro kinase assays we identified both ERK1 and ERK2 to phosphorylate NIPA with high efficiency. Mutation of either Ser 354 or Ser 359 abolished ERK-dependent NIPA phosphorylation. Inhibition of ERK1/2 activity in cell lines by specific inhibitors resulted in decreased NIPA phosphorylation at G2/M. To differentiate between phosphorylation by ERK1 and ERK2, we combined cell cycle analysis with stable expression of microRNA's targeting both isoforms. To this end NIH/3T3 cells were retrovirally transduced with microRNAs targeting ERK1 and 2 and cell cycle progression was analysed by BRDU/PI labeling. Using this approach, we are able to show that ERK2 but not ERK1 mediates NIPA phosphorylation at G2/M. Furthermore, ERK2 silencing leads to a distinct phenotype in cell cycle progression with a delay of ERK2 knockdown cells at the G2/M transition. Thus, our data indicate, that the recently described divergent functions of ERK1 and ERK2 in cell cycle regulation could be in part due to the differential ability of these kinases to phosphorylate and inactivate NIPA at G2/M. Since checkpoint proteins such as NIPA are constitutively inactivated in tumor cells ERK2 might represent an interesting target to reconstitute important cell cycle checkpoint controls in malignant cells. Disclosures:No relevant conflicts of interest to declare.

The Human Papillomavirus Type 18 E2 Protein Is a Cell Cycle-Dependent Target of the SCF Skp2 Ubiquitin Ligase

The human papillomavirus type 18 (HPV-18) E2 gene is inactivated in cervical carcinoma after integration of the viral DNA into the host cellular genome. Since E2 represses the transcription of the two viral oncogenes E6 and E7, integration which allows their strong expression is considered a major step in transformation by HPV. We show here that E2 is specifically degraded at the end of the G(1) phase in a Brd4-independent manner, implying that its regulatory functions are cell cycle dependent. Degradation of E2 occurs via the Skp1/Cullin1/F-box Skp2 (SCF(Skp2)) ubiquitin ligase, since silencing of Skp2 induces stabilization of E2. In addition, the amino-terminal domain of E2 can interact with Skp2 as shown by coimmunoprecipitation experiments. We previously showed that E2 inhibits the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, leading to accumulation of several of its substrates. We demonstrate here that Skp2, which is a known APC/C substrate in G(1), is also stabilized by E2. Therefore, by negative feedback, SCF(Skp2) activation could lead to E2 degradation and E6/E7 expression specifically in the late G(1) phase. Moreover, since the SCF(Skp2) can trigger S-phase entry and Skp2 itself is a known oncogene, we believe that E2-mediated accumulation of Skp2, together with E2 degradation leading to putative release of E6 and E7 inhibition, could induce premature S-phase entry in HPV-infected cells, pointing to a direct role of E2 in the early steps of HPV-mediated transformation.

Cyclin A/cdk2 Regulates Adenomatous Polyposis Coli-dependent Mitotic Spindle Anchoring

Mutations in adenomatous polyposis coli (APC) protein is a major contributor to tumor initiation and progression in several tumor types. These mutations affect APC function in the Wnt-beta-catenin signaling and influence mitotic spindle anchoring to the cell cortex and orientation. Here we report that the mitotic anchoring and orientation function of APC is regulated by cyclin A/cdk2. Knockdown of cyclin A and inhibition of cdk2 resulted in cells arrested in mitosis with activation of the spindle assembly checkpoint. The mitotic spindle was unable to form stable attachments to the cell cortex, and this resulted in the spindles failing to locate to the central position in the cells and undergo dramatic rotation. We have demonstrated that cyclin A/cdk2 specifically associates with APC in late G2 phase and phosphorylates it at Ser-1360, located in the mutation cluster region of APC. Mutation of APC Ser-1360 to Ala results in identical off-centered mitotic spindles. Thus, this cyclin A/cdk2-dependent phosphorylation of APC affects astral microtubule attachment to the cortical surface in mitosis.

A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication

Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.

TGFβ prevents proteasomal degradation of the cyclin-dependent kinase inhibitor p27kip1 for cell cycle arrest

TGF-β mediates cell cycle arrest in late G1 phase with a simultaneous peak in the levels of the cyclin-dependent kinase inhibitor, p27kip1 (p27). In this report, we show that whereas p27 resides in the cytoplasm in the endometrial carcinoma (ECA) cell line HEC-1A, TGF-β increases the total levels and translocation of p27 into the nucleus. Concomitantly, TGF-β activates the transcription factors Smad2 and Smad3, inhibits proliferation, and blocks Cdk2 activity; all these events are blocked by an inhibitor of TβRI serine kinase activity (SD208). In addition, we show that inhibiting p27 transcription with a specific siRNA completely blocks TGF-β-mediated growth inhibition in these cells. These data suggest that TGF-β inhibits cellular proliferation by increasing p27 levels through Smad2/3 signaling in HEC-1A cells. We further show that TGF-β decreases the levels of components of the SCFSkp2 targeting complex for ubiquitin-mediated degradation of p27 in proteasomes, at the protein but not the mRNA level. Therefore, TGF-β accumulates nuclear p27 by preventing its degradation to enable G1 arrest in HEC-1A cells. Importantly, these data suggest a novel mechanism for TGF-β/Smad mediated growth inhibition that might be inoperable in the numerous human cancers demonstrating early dysregulated TGF¬-β signaling and loss of growth inhibition. The TGF-β/p27 axis might provide novel therapeutic targets for cancer.

Factors Affecting the Diversity of DNA Replication Licensing Control in Eukaryotes

Replication of eukaryotic genomes is limited to once per cell cycle, by a two-step mechanism. DNA replication origins are first "licensed" during G1 phase by loading of an inactive DNA helicase (Mcm2-7) into pre-replicative complexes (pre-RCs). Initiation then occurs during S phase, triggered by cyclin-dependent kinases (CDKs), which promote recruitment of proteins required for helicase activation and replisome assembly. CDKs and the anaphase promoting complex/cyclosome (APC/C) restrict licensing to G1 phase by directly and indirectly regulating pre-RC components, including ORC, Cdc6, Cdt1, and Mcm2-7. Despite the fundamental importance of licensing regulation, the mechanisms by which pre-RC components are regulated differ widely across Eukarya. Here we show that even within the genus Saccharomyces, Cdc6 is regulated differently in different species. We propose that two factors contribute to the rapid evolution of licensing regulation. The first is redundancy: eliminating any single pre-RC-regulatory mechanism has very little affect on viability. The second is interchangeability: we show that regulatory mechanisms can be swapped between pre-RC components without compromising the block to re-replication. These experiments provide a framework for understanding the diversity of licensing regulation in eukaryotes and provide new tools for manipulating the chromosome-replication cycle.

Effect of 5-fluorouracil and 5-fluorouridine on metaphase chromosome structure in human lymphoid cells.

5-fluorouracil (5 FUra) and 5-fluorouridine (FUrd) both inhibit DNA synthesis in eucaryotic cells in vivo and in vitro due to inhibition of thymidylate synthetase. In addition to this effect both agents can be incorporated into RNA. Both agents demonstrate antitumour activity, but only FUra is used clinically due to the high general toxicity of FUrd, which partly seems to be due to interference with posttranscriptional modifications of tRNA and rRNA, and partly to a direct inhibition of RNA synthesis. When added to human lymphoid G2-phase cells in submitostatic concentrations, FUrd induces a structural alteration of the chromosomes in the treated cells, while a similar effect could not be observed after FUra treatment. Thus, it is suggested that the structural alteration of the chromosomes after FUrd treatment is due to a partial inhibition of RNA synthesis in late G2 phase.

Cdk1/Cdc28-Dependent Activation of the Major Triacylglycerol Lipase Tgl4 in Yeast Links Lipolysis to Cell-Cycle Progression

Triacylglycerols (TGs) serve essential cellular functions as reservoirs for energy substrates (fatty acids) and membrane lipid precursors (diacylglycerols and fatty acids). Here we show that the major yeast TG lipase Tgl4, the functional ortholog of murine adipose TG lipase ATGL, is phosphorylated and activated by cyclin-dependent kinase 1 (Cdk1/Cdc28). Phospho-Tgl4-catalyzed lipolysis contributes to early bud formation in late G1 phase of the cell cycle. Conversely, lack of lipolysis delays bud formation and cell-cycle progression. In the absence of beta-oxidation, lipolysis-derived metabolites are thus required to support cellular growth. TG homeostasis is the only metabolic process identified as yet that is directly regulated by Cdk1/Cdc28-dependent phosphorylation of key anabolic and catabolic enzymes, highlighting the importance of FA storage and mobilization during the cell cycle. Our data provide evidence for a direct link between cell-cycle-regulatory kinases and TG degradation and suggest a general mechanism for coordinating membrane synthesis with cell-cycle progression.

M Phase-Specific Phosphorylation of Histone H1.5 at Threonine 10 by GSK-3

H1 histones are progressively phosphorylated during the cell cycle. The number of phosphorylated sites is zero to three in late S phase and increases to five or six in late G2 phase and M phase. It is assumed that this phosphorylation modulates chromatin condensation and decondensation, but its specific role remains unclear. Recently, it was shown that the somatic H1 histone subtype H1.5 becomes pentaphosphorylated during mitosis, with phosphorylated threonine 10 being the last site to be phosphorylated. We have generated an antiserum specific for human H1.5 phosphorylated at threonine 10. Immunofluorescence labeling of HeLa cells with this antiserum revealed that the phosphorylation at this site appears in prometaphase and disappears in telophase, and that this hyperphosphorylated form of H1.5 is mainly chromatin-bound in metaphase when chromatin condensation is maximal. In search of the kinase responsible for the phosphorylation at this site, we found that threonine 10 of H1.5 can be phosphorylated by glycogen synthase kinase-3 in vitro, but not by cyclin-dependent kinase 1/cyclin B and cyclin-dependent kinase 5/p35, respectively. Furthermore, addition of specific glycogen synthase kinase-3 inhibitors led to a reduction in phosphorylation at this site both in vivo and in vitro.

Zona Occludens-2 Inhibits Cyclin D1 Expression and Cell Proliferation and Exhibits Changes in Localization along the Cell Cycle

Here, we have studied the effect of the tight junction protein zona occludens (ZO)-2 on cyclin D1 (CD1) protein expression. CD1 is essential for cell progression through the G1 phase of the cell cycle. We have found that in cultures of synchronized Madin-Darby canine kidney cells, ZO-2 inhibits cell proliferation at G0/G1 and decreases CD1 protein level. These effects occur in response to a diminished CD1 translation and an augmented CD1 degradation at the proteosome triggered by ZO-2. ZO-2 overexpression decreases the amount of Glycogen synthase kinase-3beta phosphorylated at Ser9 and represses beta-catenin target gene expression. We have also explored the expression of ZO-2 through the cell cycle and demonstrate that ZO-2 enters the nucleus at the late G1 phase and leaves the nucleus when the cell is in mitosis. These results thus explain why in confluent quiescent epithelia ZO-2 is absent from the nucleus and localizes at the cellular borders, whereas in sparse proliferating cultures ZO-2 is conspicuously present at the nucleus.