Checkpoint controls that couple mitosis to completion of DNA replication.

Cells treated with inhibitors of DNA synthesis do not normally enter mitosis. Incompletely replicated DNA apparently activates a regulatory mechanism that prevents activation of the mitotic inducer M-phase kinase by controlling the dephosphorylation of a critical tyrosine residue in the active site of the kinase. The control system may also target a second mitotic inducer, possibly the NIMA protein kinase. Unreplicated DNA may be detected and signalled by a complex of RCC1, a DNA-binding protein, and Ran, a Ras-related protein. This article reviews these recent developments and discusses the possibility that the control system also operates in the normal cell cycle, to ensure that mitosis strictly follows S phase.

The topoisomerase II inhibitor VM-26 induces marked changes in histone H1 kinase activity, histones H1 and H3 phosphorylation, and chromosome condensation in G2 phase and mitotic BHK cells.

We have examined the effects of topoisomerase inhibitors on the phosphorylation of histones in chromatin during the G2 and the M phases of the cell cycle. Throughout the G2 phase of BHK cells, addition of the topoisomerase II inhibitor VM-26 prevented histone H1 phosphorylation, accompanied by the inhibition of intracellular histone H1 kinase activity. However, VM-26 had no inhibitory effect on the activity of the kinase in vitro, suggesting an indirect influence on histone H1 kinase activity. Entry into mitosis was also prevented, as monitored by the absence of nuclear lamina depolymerization, chromosome condensation, and histone H3 phosphorylation. In contrast, the topoisomerase I inhibitor, camptothecin, inhibited histone H1 phosphorylation and entry into mitosis only when applied at early G2. In cells that were arrested in mitosis, VM-26 induced dephosphorylation of histones H1 and H3, DNA breaks, and partial chromosome decondensation. These changes in chromatin parameters probably reverse the process of chromosome condensation, unfolding condensed regions to permit the repair of strand breaks in the DNA that were induced by VM-26. The involvement of growth-associated histone H1 kinase in these processes raises the possibility that the cell detects breaks in the DNA through their effects on the state of DNA supercoiling in constrained domains or loops. It would appear that histone H1 kinase and topoisomerase II work coordinately in both chromosome condensation and decondensation, and that this process participates in the VM-26-induced G2 arrest of the cell.

Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation.

The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.

Food restriction induces long-lasting recovery of spatial memory deficits following global ischemia in delayed matching and non-matching-to-sample radial arm maze tasks.

Food restriction has been shown to be beneficial for a number of brain processes. In the current study, we characterized the impact of food restriction on hippocampal damage 70 days following ischemia. We assessed memory and cognitive flexibility of ad libitum fed (AL) and food-restricted (FR) animals using complex delayed non-matching- and matching-to-sample tasks in the radial arm maze. Our findings demonstrate that food restriction led to significant improvement of ischemia-induced memory impairments. FR ischemic animals rapidly reached comparable performance as both AL and FR sham animals in delayed-non-matching (win-shift) and matching (win-stay) radial arm maze tasks. They also made considerably fewer microchoices in the retention trials than AL ischemic animals. In contrast, AL ischemic rats showed persistent spatial memory impairments in the same paradigms. Assessment of basal and stress-induced corticosterone (CORT) secretion revealed no significant differences in baseline levels in AL and FR rats prior to or following global ischemia. However, FR animals showed a more pronounced attenuation of CORT secretion 45 min following restraint. Both FR and AL ischemic rats had comparable cell loss within CA1 and CA3 subfields of Ammon's horn (CA1 and CA3) at 70 days following reperfusion, although a trend toward increased CA3 cell survival was observed in FR ischemic rats. The functional sparing in the FR ischemic animals in the face of equivalent hippocampal cell loss suggests that food restriction somehow enhanced the efficacy of remaining hippocampal or extrahippocampal neurons following ischemia. In the current study, this phenomenon was not associated with diet- and or ischemia-related alterations of vesicular glutamate transporter 1 expression in various hippocampal regions although lower vesicular GABA transporter immunostaining was present in the CA1 stratum oriens and the CA3 stratum radiatum in FR sham and ischemic rats.

Antitumor drug fostriecin inhibits the mitotic entry checkpoint and protein phosphatases 1 and 2A.

In most eukaryotic cells, entry into mitosis is tightly controlled and requires completely replicated and undamaged DNA. We show that the antitumor drug, fostricin, interferes with this control; it induces cycling cells to enter mitosis prematurely, and it can overcome the mitotic entry checkpoint, forcing into mitosis cells that were arrested in the division cycle by treatment with the DNA replication inhibitor aphidicolin or with the DNA-damaging agents camptothecin and teniposide. This effect was observed in all rodent, simian, and human cell lines tested. Fostriecin also hampers progression through the later stages of mitosis as determined by the absence of normal half-spindles, anaphase figures, and telophase figures. The only previously known target for fostriecin is topoisomerase II, which is inhibited in vitro with a 50% inhibitory concentration of 40 microM (T. J. Boritzki, T. S. Wolfard, J. A. Besserer, R. C. Jackson, and D. W. Fry. Inhibition of type II topoisomerase by fostriecin. Biochem. Pharmacol., 37: 4063-4068, 1988). We show that fostriecin is a more potent inhibitor of protein phosphatase 1, with a 50% inhibitory concentration of 4 microM and protein phosphatase 2A, with a 50% inhibitory concentration of 40 nM. Inhibition of the mitotic entry checkpoint and inhibition of protein phosphatases are novel properties for antitumor drugs with potential or proven therapeutic value.

Inhibition of tumor cell invasion and angiogenesis by motuporamines.

Tissue invasion is an important determinant of angiogenesis and metastasis and constitutes an attractive target for cancer therapy. We have developed an assay to identify agents that inhibit invasion by mechanisms other than inhibition of cell attachment or cytotoxicity. A screen of marine sponge extracts identified motuporamines as micromolar inhibitors of invasion of basement membrane gels by MDA-231 breast carcinoma, PC-3 prostate carcinoma, and U-87 and U-251 glioma cells. Motuporamine C inhibits cell migration in monolayer cultures and impairs actin-mediated membrane ruffling at the leading edge of lamellae. Motuporamine C also reduces beta1-integrin activation, raising the possibility that it interferes with "inside-out" signaling to integrins. In addition, motuporamine C inhibits angiogenesis in an in vitro sprouting assay with human endothelial cells and an in vivo chick chorioallantoic membrane assay. The motuporamines show little or no toxicity or inhibition of cell proliferation, and they are structurally simple and easy to synthesize, making them attractive drug candidates.

Cell-based screen for antimitotic agents and identification of analogues of rhizoxin, eleutherobin, and paclitaxel in natural extracts.

We describe a cell-based assay for antimitotic compounds that is suitable for drug discovery and for quantitative determination of antimitotic activity. In the assay, cells arrested in mitosis as a result of exposure to antimitotic agents in pure form or in crude natural extracts are detected by ELISA using the monoclonal antibody TG-3. The assay was used to screen >24,000 extracts of marine microorganisms and invertebrates and terrestrial plants and to guide the purification of active compounds from 5 of 119 positive extracts. A new rhizoxin analogue was found in a Pseudomonas species, six new eleutherobin analogues were identified from the octocoral Erythropodium caribaeorum, and two paclitaxel analogues were found in the stem bark of the tree Ilex macrophylla. The assay was also used for quantitative comparison of the antimitotic activity of different analogues. It revealed the importance of the C-11 to C-13 segment of the diterpene core of eleutherobin for its antimitotic activity. The identification of antimitotic compounds in very low abundance and their high (0.5%) occurrence in natural extracts indicates that drug discovery efforts using this cell-based assay may lead to the identification of structurally novel antimitotic agents.

High-throughput assay for G2 checkpoint inhibitors and identification of the structurally novel compound isogranulatimide.

Treatment of cancer cells lacking p53 function with G2 checkpoint inhibitors sensitizes them to the toxic effects of DNA damage and has been proposed as a strategy for cancer therapy. However, few inhibitors are known, and they have been found serendipitously. We report the development of a G2 checkpoint inhibition assay that is suitable for high-throughput screening and its application to a screen of 1300 natural extracts. We present the isolation of a new G2 checkpoint inhibitor, the structurally novel compound isogranulatimide. In combination with gamma-irradiation, isogranulatimide selectively kills MCF-7 cells lacking p53 function.

Newly synthesized secretory proteins from pig pancreas are not released from a homogeneous granule compartment.

The pancreatic secretion of anesthetized pigs was collected by cannulation after pulse labeling with [3H]leucine. Collection at 5 min intervals started immediately post-pulse labeling up to 85 min. The volume, the protein content and the trichloroacetic acid-precipitable radioactivity of the juice were measured. The specific radioactivity of the secretory proteins was compared to that of a zymogen granule fraction isolated from the same animal. The latter was very much higher. Caerulein stimulation for 5 min at 80 min post-pulse caused a sharp drop in the specific activity of secretory proteins in the juice, to a level lower than that of the zymogen granule content. These data support the concept of more than one pool of secretory proteins in the pancreas and are incompatible with the concept that secretory proteins derive from an homogeneous granule compartment in a functionally homogeneous population of cells. To explain our results the hypothesis of a second intracellular route for the secretory proteins in proposed.

Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms.

PURPOSE: To define the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of the novel protein kinase inhibitor, UCN-01 (7-hydroxystaurosporine), administered as a 72-hour continuous intravenous infusion (CIV). PATIENTS AND METHODS: Forty-seven patients with refractory neoplasms received UCN-01 during this phase I trial. Total, free plasma, and salivary concentrations were determined; the latter were used to address the influence of plasma protein binding on peripheral tissue distribution. The phosphorylation state of the protein kinase C (PKC) substrate alpha-adducin and the abrogation of DNA damage checkpoint also were assessed. RESULTS: The recommended phase II dose of UCN-01 as a 72-hour CIV is 42.5 mg/m(2)/d for 3 days. Avid plasma protein binding of UCN-01, as measured during the trial, dictated a change in dose escalation and administration schedules. Therefore, nine patients received drug on the initial 2-week schedule, and 38 received drug on the recommended 4-week schedule. DLTs at 53 mg/m(2)/d for 3 days included hyperglycemia with resultant metabolic acidosis, pulmonary dysfunction, nausea, vomiting, and hypotension. Pharmacokinetic determinations at the recommended dose of 42.5 mg/m(2)/d for 3 days included mean total plasma concentration of 36.4 microM (terminal elimination half-life range, 447 to 1176 hours), steady-state volume of distribution of 9.3 to 14.2 L, and clearances of 0.005 to 0.033 L/h. The mean total salivary concentration was 111 nmol/L of UCN-01. One partial response was observed in a patient with melanoma, and one protracted period ( > 2.5 years) of disease stability was observed in a patient with alk-positive anaplastic large-cell lymphoma. Preliminary evidence suggests UCN-01 modulation of both PKC substrate phosphorylation and the DNA damage-related G(2) checkpoint. CONCLUSION: UCN-01 can be administered safely as an initial 72-hour CIV with subsequent monthly doses administered as 36-hour infusions.

Chromosomal loop/nuclear matrix organization of transcriptionally active and inactive RNA polymerases in HeLa nuclei.

The relative distribution of transcriptionally active and inactive RNA polymerases I and II between the nuclear matrix/scaffold and chromosomal loops of HeLa cells was determined. Total RNA polymerase was assessed by immunoblotting and transcribing RNA polymerase by a photoaffinity labeling technique in isolated nuclei. Nuclear matrix/scaffold was isolated by three methods using high-salt, intermediate-salt or low-salt extraction. The distribution of RNA polymerases I and II were very similar within each of the methods, but considerable differences in distributions were found between the different preparation methods. Either intermediate-salt or high-salt treatment of DNase I-digested nuclei showed significant association of RNA polymerases with the nuclear matrix. However, intermediate-salt followed by high-salt treatment released all transcribing and non-transcribing RNA polymerases. Nuclear scaffolds isolated with lithium diiodosalicylate (low-salt) contained very little of the RNA polymerases. This treatment, however, caused the dissociation of RNA polymerase II transcription complexes. These results show unambiguously that RNA polymerases, both in their active and inactive forms, are not nuclear matrix proteins. The data support models in which the transcriptional machinery moves around DNA loops during transcription.

Distortion of the DNA double helix by RAP1 at silencers and multiple telomeric binding sites.

Repressor Activator Protein 1 (RAP1) is an essential nuclear protein of the yeast Saccharomyces cerevisiae that recognizes a 13 base-pair (bp) consensus sequence found in numerous upstream activating sequences, at the silencers of transcriptionally repressed mating-type genes, and in telomeric tracts, called (C1-3 A) repeats. RAP1 has been shown to influence transcriptional activation, transcriptional repression, telomere length, circular plasmid segregation and meiotic recombination in vivo. We have studied the structure of the protein-DNA complex reconstituted in vitro with highly purified RAP1, by using DNase I and chemical footprinting. Both full-length RAP1 and its minimal DNA-binding domain of roughly 30 kDa, induce a distortion within the 13 bp recognition site, as demonstrated by reactivity to KMnO4 primarily at nucleotides 8 and 10 in the binding consensus Rc/AAYCCRYNCAYY. Dimethylsulphate reactivity shows that RAP1 binding does not create unpaired regions at its binding site, although the DNA may be locally underwound or aberrantly base-paired at the permanganate reactive nucleotides. In addition to the permanganate-sensitive distortion, the full-length RAP1, but not its DNA-binding domain, induces a bend in DNA 5' of the recognition sequence, altering the electrophoretic mobility of the protein-DNA complex. The KMnO4-reactivity has allowed a precise mapping of RAP1 molecules on telomeric DNA, revealing RAP1 sites as frequently as one per 18 bp of telomeric DNA, or potentially 20 RAP1 molecules bound per average telomeric tract of 370 bp. This suggests that RAP1 plays a major role in organizing yeast telomeres, and is consistent with recently published immunofluorescence studies showing a major fraction of RAP1 at the ends of meiotic chromosomes.

Nucleosome arrays inhibit both initiation and elongation of transcripts by bacteriophage T7 RNA polymerase.

We have examined the effects of nucleosome cores on the initiation and elongation of RNA transcripts by phage T7 RNA polymerase in vitro. A transcription template, pT207-18, was constructed containing tandemly repeated 207 base-pair (bp) nucleosome positioning sequences from a sea urchin (Lytechinus variegatus) 5 S RNA gene inserted between the T7 and SP6 transcription promoters of pGEM-3Z. Nucleosome cores were reconstituted onto supercoiled, closed circular pT207-18 DNA and double label transcription experiments were performed to determine the effects of nucleosome cores on the initiation and elongation of transcripts by T7 RNA polymerase. Both transcript initiation and elongation were inhibited, the extent of the inhibition being directly proportional to the number of nucleosome cores reconstituted onto the pT207-18 DNA templates. Time course transcription experiments indicated that nucleosome cores caused a reduction in the equilibrium length of transcripts and not mere retardation of elongation rates. Continuous regularly spaced linear arrays of nucleosomes were obtained by digesting reconstituted nucleosomel pT207-18 templates with DraI, for which a unique restriction site lies within the nucleosome positioning region of the 207 bp 5 S rDNA repeat sequence. After in vitro transcription with T7 RNA polymerase an RNA ladder with 207 nucleotide spacing was obtained, indicating that transcription can occur through continuous arrays of positioned nucleosome cores. It is demonstrated that nucleosome cores partially inhibit the elongation of transcripts by T7 RNA polymerase, while allowing passage of the transcribing polymerase through each nucleosome core at an upper limit efficiency of 85%. Hence, complete transcripts are produced with high efficiency from short nucleosomal templates, while the production of full-length transcripts from long nucleosomal arrays is relatively inefficient. The results indicate that nucleosome cores have significant inhibitory effects in vitro not only on transcription initiation but on transcription elongation as well, and that special mechanisms may exist to overcome these inhibitory effects in vivo.

Isolation of zymogen granules from rat pancreas and characterization of their membrane proteins.

A zymogen granule fraction has been isolated from rat pancreas, and its purity has been assessed by biochemical and morphological criteria. Specific activities of two marker enzymes, amylase and chymotrypsin, are increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction is devoid of detectable RNA, DNA and 5'-nucleotidase, glucose-6-phosphatase, and cytochrome c oxidase activities. Electron micrographs confirm the absence of mitochondria, lysosomes, and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase, and low levels of acid phosphatase. Membrane proteins were solubilized by urea-Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Isoelectric point and molecular weight of each protein band were determined.

The origin of the zymogen granule membrane of the pancreatic acinar cell as examined by ultrastructural cytochemistry of acid phosphatase, thiamine pyrophosphatase, and ATP-diphosphohydrolase activities.

Cytochemical distributions of acid phosphatase, thiamine pyrophosphatase, and ATP-diphosphohydrolase activities have been examined on thin sections of rat pancreas and on isolated zymogen-granule membranes. Acid phosphatase was found in the rigid lamellae separated from the Golgi stacked cisternae, in condensing vacuoles, and in the trans-saccules of Golgi apparatus; it was not detected in purified zymogen-granule membranes. Thiamine pyrophosphatase was detected in trans-saccules of the Golgi apparatus, in purified zymogen-granule membranes, and in the plasmalemma of the acinar cell. It was absent in condensing vacuoles. The ATP-diphosphohydrolase activity has a distribution similar to thiamine pyrophosphatase. These observations illustrate the similarity between the trans-saccules of the Golgi apparatus and the membrane of mature zymogen granules and the disparity between the latter membrane and the membrane of the condensing vacuole. They suggest that the condensing vacuole might not be the immediate precursor of the zymogen granule as commonly assumed. An alternative possibility would be that condensing vacuoles would fuse with the trans-saccule (transition) of the Golgi apparatus which in turn would form mature zymogen granules.

Inhibition of 5S RNA transcription in vitro by nucleosome cores with low or high levels of histone acetylation.

Nucleosomes exert strong inhibitory effects on gene transcription in vitro and in vivo. Since most DNA is packaged in nucleosomes, there must exist mechanisms to alleviate this inhibition during gene activation. Nucleosomes could be destabilized by histone acetylation which is strongly correlated with gene expression. We have compared the effects of nucleosomes cores with low or high levels of histone acetylation on 5S RNA transcription with Xenopus nuclear extracts in vitro. Little or no difference was observed over a range of 1 to 15 nucleosome cores per plasmid template. This result suggests that nucleosomal DNA is not more accessible to transcription factors and to the transcription machinery in acetylated nucleosomes.

The hydra head activator in human blood circulation. Degradation of the synthetic peptide by plasma angiotensin-converting enzyme.

Using methanol extraction combined with HPLC and a new radioimmunoassay, the peptide head activator was detected in human plasma at a concentration of 20-100 fmol/ml. Synthetic head activator incubated with plasma was degraded with a half-life of 7 min. Analysis of sites of enzymatic cleavage and inhibition by captopril showed a major involvement of angiotensin-converting enzyme in this process. Endogenous head activator, on the other hand, was not appreciably degraded upon incubation of plasma in vitro. These findings raise the possibility that the endogenous peptide could bind to a protective carrier molecule and reach potential target tissues via the blood circulation.

Characterization of topoisomerase II-DNA interaction and identification of a DNA-binding domain by ultraviolet laser crosslinking.

We have used ultraviolet laser crosslinking to characterize the DNA-binding properties of highly purified yeast topoisomerase II in the absence of ATP. A single 5 ns, 20 mJ pulse of 266 nm light produced optimal crosslinking to a short DNA duplex, with an efficiency of 0.25%. An equilibrium binding constant (Keq) of 1.2 +/- 0.5 x 10(8) M(-1) was determined from kinetic analysis. Topoisomerase II showed highest affinity for supercoiled DNA. Limited proteolysis of crosslinked topoisomerase II-DNA complexes showed a site of crosslinking to be within a 29-kDa fragment with Leu-681 at its amino-terminal end. This region contains the active Tyr-783 and is homologous to the amino-terminal region of the DNA-binding bacterial gyrase GyrA subunit, suggesting a conserved DNA-binding mechanism.

Structure-activity relationships for G2 checkpoint inhibition by caffeine analogs.

Caffeine inhibits the G2 checkpoint activated by DNA damage and enhances the toxicity of DNA-damaging agents towards p53-defective cancer cells. The relationship between structure and G2 checkpoint inhibition was determined for 56 caffeine analogs. Replacement of the methyl group at position 3 or 7 resulted in loss of activity, while replacement at position 1 by ethyl or propyl increased activity slightly. 8-Substituted caffeines retained activity, but were relatively insoluble. The structure-activity profile did not resemble those for other known pharmacological activities of caffeine. The active analogs also potentiated the killing of p53-defective cells by ionizing radiation, but none was as effective as caffeine.