Cell cycle-dependent establishment of a late replication program

DNA replication origins in chromosomes of eukaryotes are activated according to a temporal program. In the yeast Saccharomyces cerevisiae, activation of origins in early S phase appears to be a default state. However, cis-acting elements such as telomeres can delay origin activation until late S phase. Site-specific recombination was used to separate origin from telomere in vivo, thereby demonstrating that the signal for late activation is established between mitosis and START in the subsequent G1 phase. Once set, the signal can persist through the next S phase in the absence of the telomere. Establishment of the temporal program and of initiation competence of origins may be coincident events.     

Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus

We reexamined 21 patients with congenital pseudarthrosis of the leg (congenital pseudoarthrosis of the tibia; CPT) associated with neurofibromatosis-1 (NF-1), > or =2 years after the termination of treatment, for a statistical study of the results obtained by using Ilizarov's external fixator. Of the 21 tibias operated on, 17 consolidated after the first treatment, whereas four did not. Of the 17 consolidated tibias, four refractured and were retreated by using a variety of methods. Only one healed. At follow-up, which occurred > or =2 years after the removal of the fixator, the results were nine consolidations without deformities or with shortening <2 cm, five consolidations with axial deviation, and seven nonconsolidations. The statistically significant results were that (a) patients who were aged 5 years or older at operation had better results, and (b) the assembly II (resection of CPT stumps and their short-term compression possibly associated with corticotomy or epiphyseal distraction to correct limb discrepancy) gave better final results compared with the other device assemblies. We conclude that treatment with Ilizarov's fixator allows (a) a good percentage of healing over time (66.7%), especially in cases of normotrophic and cystic CPT; (b) further operations with or without the fixator to correct secondary or residual axial deviation; and (c) correction of limb discrepancy. This treatment avoids risking injury to the healthy contralateral leg. Additionally, for treatments that do not achieve satisfactory results, other treatment methods are not excluded. The CPT still remains a difficult problem for the orthopedic surgeon to solve.     

Cell cycle-dependent expression of estrogen receptor and effect of estrogen on proliferation of synchronized human osteoblast-like osteosarcoma cells

Dual fluoroimmunohistochemical staining of estrogen receptor (ER) and bromodeoxyuridine was performed in a human osteoblastic osteosarcoma cell line, HOS TE85 cells. ER immunoreactivity was observed preferentially in the nuclei of the cells that were bromodeoxyuridine positive. ER expression at various phases of the cell cycle was investigated in HOS TE85 cells, which were synchronized at the G1/S phase boundary by intermittent exposure to thymidine and hydroxyurea. ER immunoreactivity became detectable in the S phase, decreased in the G2/M and G1 phases, and then reappeared in the S phase of the next cell cycle. Western blot analysis also showed that ER protein exists in these cells and increases in the S phase. Moreover, Northern blot analysis demonstrated that the expression of ER messenger RNA increases in the early S phase, gradually decreases during the progress of the cell cycle, and increases again in the S phase of the subsequent cell cycle. Interestingly, 17 beta-estradiol (10(-8) M) increased cell number and [3H]thymidine incorporation into DNA in the synchronized HOS TE85 cells, whereas this effect was not observed in the nonsynchronized HOS TE85 cells. The present studies suggest that the cell cycle-dependent regulation may contribute to the heterogeneity of ER expression in osteoblastic cells.     

Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation

DNA polymerase alpha-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase alpha-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase alpha-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.     

Eucaryotic DNA primase does not prefer to synthesize primers at pyrimidine rich DNA sequences when nucleoside triphosphates are present at concentrations found in whole cells

The critical role of NTP concentration in determining where calf thymus DNA primase synthesizes a primer on a DNA template was examined. Varying the concentration of NTPs dramatically altered the template sequences at which primase synthesized primers. At the low NTP concentrations typically used for in vitro experiments (100 microM), primase greatly preferred to synthesize primers at pyrimidine rich DNA sequences. However, when the concentrations of NTPs were increased to levels typically found in whole cells, primers were now synthesized in all regions of the template. Importantly, synthesis of primers in all regions of the DNA template, not just the pyrimidine rich sequences, is the pattern of primer synthesis observed during DNA replication in whole cells. With low concentrations of NTPs (i.e., Vmax/K(M) conditions), primers are only synthesized at the most preferred synthesis sites, namely, those that are pyrimidine rich. In contrast, under conditions of high NTP concentrations, primer synthesis will occur at the first potential synthesis site to which primase binds. Now, the primase x DNA complex will be immediately converted to a primase x DNA x NTP x NTP complex that is poised for primer synthesis.     

Cell cycle-dependent duplication and bidirectional migration of SeqA-associated DNA-protein complexes in E. coli

Using immunofluorescence microscopy, we have found that SeqA protein, a regulator of replication initiation, is localized as discrete fluorescent foci in E. coli wild-type cells. Surprisingly, SeqA foci were observed also in an oriC deletion mutant. Statistical analysis revealed that a SeqA focus is localized at midcell in newborn cells. The SeqA focus is duplicated and tethered at midcell until an FtsZ ring is formed. Subsequently, these foci migrate in opposite directions toward cell quarter sites and remain tethered there until the cell divides. The cell cycle-dependent bidirectional migration of SeqA-DNA complexes is quite different from the migration pattern of oriC Dna copies. MukB protein is required for correct localization of SeqA complexes by an unknown mechanism.     

Topoisomerase poisons activate the transcription factor NF-kappaB in ACH-2 and CEM cells

The nuclear factor kappaB (NF-kappaB) is involved in T cell activation and enhances HIV-1 gene expression. It is activated in response to numerous stimuli, including oxidative stress. Oxidative stress damages membrane lipids, proteins and nucleic acids. We have shown previously that oxidative DNA damage generated by photosensitization could trigger activation of NF-kappaB. We now show that a series of topoisomerase poisons (actinomycin D, camptothecin, daunomycin and etoposide) also activate NF-kappaB (NFKB1/RelA dimer) in ACH-2 and CEM cells. This activation is inhibited by pyrrolidine dithiocarbamate. In ACH-2 cells latently infected by HIV-1, camptothecin, daunomycin and etoposide are able to enhance virus production. Since topoisomerase poisons cause the formation of single- and double-strand breaks in DNA, these lesions might be capable of triggering NF-kappaB activation. Indeed, DNA damaging agents generating adducts (trans-platin and 4-nitroquinoline 1-oxide) and/or crosslinks in DNA (cisplatin and mitomycin C) do not or only weakly activate NF-kappaB in T cell lines.     

Influence of age on enzyme activities of pyrimidine metabolism in the chicken heart

We and others found that cardioprotection is acquired not only soon after, but also 24 h after ischemic preconditioning in canine and rabbit myocardial infarction models (second window of protection). However, a second window phenomenon against myocardial infarction was dependent on species limitations and has not been observed in porcine hearts. In this study, we examined whether the "second window of protection" against myocardial infarction is observed in the rat heart. In the ischemic preconditioning (IP) group, the left main coronary artery (LCA) of rats was occluded four times for 3 min. each separated by reperfusion for 10 min. After 0, 3, and 24 h, the rats were subjected to a 20-min LCA occlusion followed by 48-h reperfusion. At 0 and 24 h after IP, infarct size and the incidence of ventricular fibrillation (VF) during ischemia were significantly reduced compared with corresponding sham-operated groups without preconditioning. After 3 h of IP, there were no differences either in the incidence of VF during ischemia or in infarct size. Manganese superoxide dismutase (Mn-SOD) content in ischemic (LCA) region of myocardium significantly increased as compared with that of sham-operated rats 24 h after IP. Treatment with N-2-mercaptopropionyl glycine, an antioxidant and a hydroxyl radical scavenger, during IP abolished the early-phase (0 h after IP) and late-phase (24 h after IP) cardioprotection and the corresponding late increase in Mn-SOD content. These results indicate that a "second window of protection" against myocardial infarction also exists in rat hearts and the induction of an intrinsic scavenger, Mn-SOD, via free radical production during IP may be important in the second window of protection.     

Roles of nucleoside triphosphates in microtubule assembly

Depolymerization of microtubules in the ATP-reassembly buffer permitted the preparation of GDPETNGTP. Incubation of this tubulin fraction at 35 degrees with ATP induced the phosphorylation of E-site GDP into GTP, which was then dephosphorylated during microtubule assembly. Incubation of GDPETNGTP with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40] also induced polymerization. Depolymerization of microtubules in the GTP-reassembly buffer yielded GTPETNGTP, which was capable of polymerizing into microtubules even in the absence of free GTP. In the presence of 4 M glycerol, GDPETNGTP assembled into microtubules with no change in the bound nucleotides.     

Cell cycle-dependent cytotoxicity, G2/M phase arrest, and disruption of p34cdc2/cyclin B1 activity induced by doxorubicin in synchronized P388 cells

We studied the effect of doxorubicin (Dox) on cell cycle progression and its correlation with DNA damage and cytotoxicity in p53-mutant P388 cells. P388 cells synchronized in S and G2/M phases were > 3-fold more sensitive to Dox than were cells in G1 phase (Dox ID50 = 0.50 +/- 0.16 microM in cells synchronized in S phase versus 1.64 +/- 0.12 microM in asynchronized cells; drug exposure, 1 hr). Treatment of synchronized cells in early S phase with 1 microM Dox (2 x ID50) for 1 hr induced a marked cell arrest at G2/M phase at 6-12 hr after drug incubation. We then studied the effect of Dox on the p34cdc2/cyclin B1 complex because it plays a key role in regulating G2/M phase transition. In untreated control P388 cells, p34cdc2 kinase localizes in the nucleus and cytoplasms, particularly in the centrosomes, and p34cdc2 kinase activity is dependent on cell cycle progression, with the enzyme activity increasing steadily from G1/S to G2/M and markedly declining thereafter. Treatment of synchronized P388 cells in early S phase with 1 microM Dox for 1 hr did not affect the pattern of subcellular distribution of the enzyme but completely abrogated its function for > or = 10 hr. In a cell-free system, Dox did not inhibit p34cdc2 kinase activity, indicating that is has no direct effect on the enzyme function. In whole cells, Dox treatment prevented p34cdc2 kinase dephosphorylation without altering its synthesis, and this effect was due to neither down-regulation of cdc25C nor inhibition of protein-tyrosine phosphatase activity. In contrast, Dox treatment was found to induced cyclin B1 accumulation as a result of stimulating its synthesis and inhibiting its degradation. A good correlation was found between extent of DNA double-strand breaks and p34cdc2 kinase activity inhibition. Our results suggest that anthracycline-induced cytotoxicity is cell cycle dependent and is mediated, at least in part, by disturbance of the regulation of p34cdc2/cyclin B1 complex, thus leading to G2/M phase arrest.     

Amino acid metabolism of myeloma cells in culture

The growth of myeloma cells in Leibovitz medium supplemented with 20% serum was limited by the depletion of glutamine. A simple modification of the Leibovitz medium by increasing the concentrations of glutamine, lysine, isoleucine, leucine, sodium pyruvate, galactose, and vitamins resulted in over 100% increase in cell growth yield. The total myeloma protein produced by the cells was increased by approximately 90% in modified Leibovitz media. Analysis of spent culture media for 19 amino acids showed that the concentrations of 8 amino acids were reduced; those of 5 amino acids were increased and the other 6 did not change significantly.     

Uptake and metabolism of lipoprotein-X in mesangial cells

An HPLC method developed to detect in a single run both atenolol and chlorthalidone, extracted from plasma, using two detectors (UV for chlorthalidone and fluorometric for atenolol) connected in series, is described. The drugs were separated on an ODS column at room temperature using a 0.05 M sodium dodecyl sulphate in phosphate buffer (pH 5.8)-n-propanol (95:5, v/v) solution, delivered at a flow-rate of 1.3 ml/min. Having ascertained the sensitivity (10 ng/ml of both drugs) and the intra-day reproducibility (pre-study validation), the reliability of the method was verified by inter-day assays (within-study validation) carried out during the analysis of plasma samples collected from healthy volunteers after single-dose treatment with atenolol+chlorthalidone tablets (pharmaceutical preparations containing 100+25 mg and 50+12.5 mg of the two drugs, respectively).