Reduction in the radiation-induced late S phase and G2 blocks in HL-60 cell populations by amiloride, an efficient inhibitor of the Na+/H+ transporter.

Recent investigations that showed that amiloride delayed or inhibited apoptosis indicated it might also attenuate cell cycle checkpoints activated by ionizing radiation. In this report, single- and dual-parameter flow cytometry were used to investigate the effects of amiloride on cell cycle progression, and the effectiveness of amiloride to attenuate the S and G2 phase checkpoint responses induced by 2.5, 5.0, and 7.5 Gy of gamma radiation. The late S-phase delay, noted at 8 h following irradiation, and a radiation-induced G2 block, which was maximum at 16 h after irradiation, were both significantly reduced in amiloride-treated samples. Attenuation of the radiation-induced late S phase and G2 blocks resulted in cell division without apparent apoptosis or necrosis over a 24-h period. Results presented indicate that amiloride reduces the radiation-induced G2 block in HL-60 cell populations almost equally well as caffeine and to a greater extent than staurosporine. Immunofluorescent detection and quantitation of cyclin B1 expression demonstrated that amiloride only significantly reduced cyclin B1 expression following 5.0 Gy, when there was a notable induction of a significant G2 delay, followed by a relatively rapid recovery in cycling potential. The results suggest that amiloride affects the radiation-triggered signaling cascades to alter the kinase activity of proteins associated with mitotic progression, particularly the cyclin B1-p34cdc2 complex. Alternatively, alterations in intracellular ion concentrations induced by amiloride may lead to changes in Ca2+-dependent signaling cascades and thereby decrease the radiation-mediated cell cycle perturbations.

Requirements for p53 and the ATM gene product in the regulation of G1/S and S phase checkpoints.

We investigated the requirements for protein p53 and the ATM gene product in radiation-induced inhibition of DNA synthesis and regulation of the cyclin E/ and cyclin A/cyclin dependent kinases (Cdks). Wild type (WT) mouse lung fibroblasts (MLFs), p53(-/-) knock-out MLFs, normal human skin fibroblasts (HSF-55), and human AT skin fibroblasts (GM02052) were used in the investigations. The absence of p53 had no significant effect on the inhibition or recovery of DNA synthesis throughout the S phase, as determined from BrdU labeling and flow cytometry, or the rapid inhibition of cyclin A/Cdks. Gamma radiation (8 Gy) inhibited DNA synthesis and progression into G2 during the first 3 h after irradiation, and the recovery of these processes occurred at similar rates in both WT and p53(-/-) MLFs. The cyclin A/Cdks were inhibited 55-70% at 1 h after irradiation in both cell types, but p21WAF1/Cip1 levels or p21 interaction with Cdk2 did not increase in the irradiated p53(-/-) MLFs. Although p53(-/-) MLFs do not exhibit prolonged arrest at a G1 checkpoint, radiation did induce a rapid 20% reduction and small super-recovery of cyclin E/Cdk2 within 1-2 h after irradiation. Similar inhibition and recovery of cyclin E/Cdk2 previously had been associated with regulation of transient G1 delay and the inhibition of initiation at an apparent G1/S checkpoint in Chinese hamster cells. In contrast, loss of the ATM gene product abrogated transient cyclin E/Cdk2 inhibition, most inhibition of DNA synthesis and all, but a 10-15% inhibition, of the cyclin A/Cdks. The results indicate that neither p53 nor p21 is required for transient inhibition of cyclin E/Cdk2 associated with the G1/S checkpoint or for inhibition of DNA synthesis at 'checkpoints' within the S phase. Conversely, the ATM gene product appears to be essential for regulation of the G1/S checkpoint and for inhibition of DNA replication associated with the inhibition of cyclin A/Cdk2. Differential aspects of DNA synthesis inhibition among cell types are presented and discussed in the context of S phase checkpoints.

Changes in H1 content, nucleosome repeat lengths and DNA elongation under conditions of hydroxyurea treatment that reportedly facilitate gene amplification.

Depletion of histone H1, changes in nucleosome repeat lengths, and extents of DNA elongation were investigated in synchronized Chinese hamster (line CHO) cells using the general conditions of hydroxyurea treatment that appear to increase the frequency of gene amplification, i.e., synchronized cultures of G1 cells were allowed to begin to enter S phase before treatment with hydroxyurea was effected to retard DNA synthesis (Mariani, B.D. and Schimke, R.T. (1984) J. Biol. Chem. 259, 1901-1910). During the time that synchronized G1 cells begin to enter S phase, there occur considerable synchrony decay and accumulation of new DNA that increase with time before treatment with hydroxyurea is initiated. During exposure to hydroxyurea, there occur depletion of histone H1 and shortened repeat lengths for the DNA synthesized in the presence of hydroxyurea. In contrast, DNA synthesized in S phase before exposure to hydroxyurea has essentially the same repeat lengths as bulk chromatin at both the time that hydroxyurea treatment is effected and after 6 h in its presence. Sedimentation measurements indicate that the early replicating DNA undergoes considerable elongation both before and during 6 h of exposure to 0.3 mM hydroxyurea. Thus, nearly all of the early replicating DNA is elongated to greater than average replicon size under those conditions of hydroxyurea treatment that appear to favor gene amplification. Because the extents of DNA synthesis and cell cycle progression vary as functions of drug concentration, treatment times, and unknown factors (from experiment to experiment), it would appear that the parameters must be carefully monitored in each experiment if biochemical results are to be related to the position of cells in the growth cycle.

Composition and synthesis during G1 and S phase of a high mobility group-E/G component from Chinese hamster ovary cells.

A perchloric acid soluble protein from the sedimented chromatin of blended Chinese hamster ovary (line CHO) cells has been isolated by guanidine hydrochloride gradient chromatography on Bio . Rex-70 ion exchange resin. The amino acid composition of the protein (designated as CHO HMG-E/G) is similar to that of mouse HMG-E, but it differs from that of bovine HMG-14 and HMG-17 or any possible mixture of the two. CHO HMG-E/G incorporates [32P]phosphate like HMG-14 and HMG-17 class proteins from other species, but all resolvable molecular species incorporate phosphate, and the more highly-phosphorylated band migrates faster, rather than slower, than the other in acid-urea gel systems. Incorporation of [3H]lysine into HMG-E/G following release from isoleucine deprivation G1 block indicates that the protein is extensively synthesized during both the G1 and S phases of the cell cycle.

Localization and DNA sequence of a replication origin in the rhodopsin gene locus of Chinese hamster cells.

A chromosomal origin of DNA replication has been localized within the single-copy rhodopsin gene locus in Chinese hamster (line CHO) cells using two methods. In the first method, single-copy segments were identified at 3 to 15 kb intervals within approximately 75 kb (kb = 10(3) bases) of cloned genomic DNA containing the early-replicating rhodopsin gene near its middle. The cloned single-copy segments were then used as hybridization probes to quantify the replication of their corresponding genomic segments as synchronized cells progressed into S phase. In the second method, genomic DNA synthesized in vivo or in permeabilized early S phase cells was hybridized with slot-blots of the cloned single-copy DNA segments to identify the earliest replicating part of the 75 kb mapped region. The first method indicates that the earliest replicating DNA is located within a 10 kb region beginning 4 kb upstream from and extending 1 kb beyond the rhodopsin gene. The second method confirms the location in the vicinity of the rhodopsin gene and indicates that the earliest replicating region is located within or very near the 4.5 kb rhodopsin gene itself. An extended region of 12 kb that encompasses the entire early-replicating region has been sequenced for analysis and comparison with currently characterized origin regions associated with the CHO dihydrofolate reductase (dhfr) and human c-myc genes. There are several sequence similarities between the dhfr rhodopsin origin regions, including common transcription promoter consensus sequences, rodent Alu repeats with their 3'-A+T rich flanking sequences, A+T-rich yeast ARS and Drosophila SAR consensus sequences, and simple (GA)n repeats, but there are no extended regions of direct similarity. The rhodopsin gene locus is the second sequenced CHO origin region.

Association of G1/S-phase and late S-phase checkpoints with regulation of cyclin-dependent kinases in Chinese hamster ovary cells.

We investigated the time-dependent effects of 8 Gy of gamma radiation on the activities of cyclin-dependent kinases (Cdk's) and the incorporation of the thymidine analog bromodeoxyuridine (BrdU) throughout the S phase in Chinese hamster ovary (CHO) cells. The in vitro Cdk activities of immunoprecipitated cyclin E, cyclin A and Cdk2 were reduced about 30% per cell within 0.5-1 h after irradiation, but they recovered at different rates. The kinase activity of the cyclin E-Cdk2 complex recovered first and exceeded the control values by 1.5-2 h after irradiation. Cyclin A-Cdk activities began to recover at 3-4 h after irradiation, and cyclin E/A-Cdk2 activities recovered at intermediate rates. The super-recovery of cyclin E-Cdk2 coincided with the appearance of a small synchronous population of cells entering into S phase, consistent with transient G1-phase delay/recovery regulated by cyclin E-Cdk2, whereas the activities of cyclin A-Cdk's (75% cyclin A-Cdk2; 25% cyclin A-Cdc2 when inhibition was maximal) were correlated with rates of total DNA synthesis. Multivariate flow cytometry analyses of BrdU incorporation demonstrated that radiation-induced inhibition of DNA synthesis occurred predominantly within the last quarter of S phase and that the majority of the irradiated cells failed to enter G2 phase for 4-5 h. The recovery of cyclin A-Cdk activities coincided with increased levels of total DNA synthesis and BrdU incorporation into cells within the last quarter of S phase. Western blot analysis demonstrated that levels of Waf1/p21 did not increase during inhibition of cyclin A-Cdk's and DNA synthesis in the irradiated p53-mutated CHO cells; however, Cdc2 and Cdk2 exhibited increased levels of phosphotyrosine. The results (1) indicate that the transient G1-phase delay or G1/S-phase checkpoint (Lee et al., Proc. Natl. Acad. Sci. USA 94, 526-531, 1997) is mediated by inhibition of cyclin E-Cdk2 and (2) point to the existence of a radiation-induced S-phase checkpoint located about 75% into S phase involving the inhibition of cyclin A-Cdk's by a p53/Waf1-independent pathway in CHO cells.

The radiation-induced S-phase checkpoint is independent of CDKN1A.

We recently demonstrated that, in response to radiation, replication is down-regulated at the level of individual origins throughout S phase of the cell cycle. Since several in vitro studies demonstrate that CDKN1A (formerly known as p21) down-regulates replication by inhibiting PCNA, and since CDKN1A can retard progression of cells through S phase in vivo, the question arises whether CDKN1A is involved in the S-phase damage-sensing pathway. In the present study we analyzed the effect of ionizing radiation on CDKN1A+/+ and CDKN1A-/- cells derived from the HCT 116 cell line. Neither progression of cells through S phase nor survival after exposure to ionizing radiation is influenced by CDKN1A status in either synchronous or asynchronous cells. These results establish that CDKN1A is not necessary for the acute S-phase damage-sensing pathway that functions to prevent firing of replication origins during S phase.

Small-bowel obstruction caused by a long-term indwelling urinary catheter.

The first known case report of a small-bowel obstruction caused by a long-term indwelling Foley catheter is presented. The balloon of the catheter passed into and obstructed the lumen of the distal ileum through a vesicoenteric fistula created by chronic irritation. With the exception of recurrent urinary-tract infections, complications of urinary catheters are rare. The patient presented a diagnostic dilemma that was solved with a preoperative computed tomographic scan.

Superior mesenteric artery aneurysm: 45 years later.

Superior mesenteric artery aneurysms (SMAAs) have been described for the past 100 years, with the first successful treatment being published in 1953 by Drs. M.E. DeBakey and D.A. Cooley. It is now 45 years after this first successful treatment, and we have a case study of SMAA that depicts the typical presentation and treatment of such an aneurysm. Our SMAA was repaired with proximal and distal ligation of the aneurysm, with intestinal viability determined by Doppler probe, fluorescein dye and repeated laparotomy in 24 hours. The patient tolerated the procedure well, and there were no postoperative sequelae. In general, SMAAs have a broad range of treatments, including vessel ligation with or without excision, revascularization with primary anastomosis, and obliterative aneurysmorrhaphy. In the future, these SMAAs and other visceral aneurysms can be treated with transluminally placed endovascular grafts with the combined efforts of the vascular surgeon and the interventional radiologist. Our article provides an overall examination of SMAAs with a review of the literature.

Chromatin structural changes in synchronized cells blocked in early S phase by sequential use of isoleucine deprivation and hydroxyurea blockade.

We have investigated the loss of histone H1 from chromatin [D'Anna, J. A., Gurley, L. R., & Tobey, R. A. (1982) Biochemistry 21, 3991-4001] and the structure of chromatin from Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation G1 block and 1 mM hydroxyurea (HU) blockade. Measurements of H1 content in the cell and histone turnover indicate that H1 is lost from the cell and that there is negligible replacement synthesis of H1 during the period of the S-phase block. As H1 is lost, chromatin appears to undergo structural change. After 10 h of HU block, the new deoxyribonucleic acid (DNA) and a portion of the old DNA have measured nucleosome repeat lengths (37 degrees C digestion) which are less than those of controls and similar to those observed by Annunziato and Seale [Annunziato, A. T., & Seale, R. L. (1982) Biochemistry 21, 5431-5438] for new immature chromatin in the absence of HU. By 24 h of HU block, nearly all of the chromatin has assumed a pseudoimmature conformation in which the nucleosome cores appear to be more closely packed along the DNA chain, but the new DNA is slightly more resistant than old DNA to attack by micrococcal nuclease. Electrophoretic analysis of nucleoprotein particles produced by micrococcal nuclease digestion of nuclei indicates that (1) the distribution of mononucleosome species changes during HU block and (2) some mononucleosome species appear to be enriched in normally minor proteins which may determine the electrophoretic mobility of the nucleoprotein particles in agarose-acrylamide gels. The results raise the possibility that (1) during the early stages of replication (or prior to the passage of the replication fork), H1 is dissociated from initiated replicons and (2) H1 does not reassociate in a concerted fashion with the H1-depleted chromatin until the replication fork has passed and, perhaps, a substantial portion of the replicon has been replicated.

Changes in histone H1 content and chromatin structure of cells blocked in early S phase by 5-fluorodeoxyuridine and aphidicolin.

We have measured changes in histone H1 content and changes in chromatin structure of Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation and blockade with 5-fluorodeoxyuridine or aphidicolin. Both the H1:core histone ratio in isolated nuclei and the H1 content of the cell are reduced 20-60%, depending on the duration of the block. The new deoxyribonucleic acid (DNA) synthesized during S-phase block has a shorter nucleosome repeat length than that of bulk chromatin, but it is nearly equally resistant as bulk DNA to attack by micrococcal nuclease. During the time that H1 content is decreasing, bulk chromatin also undergoes structural changes so that its nucleosome cores appear to be more closely packed along the DNA chain. The losses in H1 content and changes in chromatin structure are similar to those reported for cells blocked in early S phase by hydroxyurea [D'Anna, J. A., & Prentice, D. A. (1983) Biochemistry 22, 5631-5640]. The results suggest that losses of H1 and changes in chromatin structure are general events which occur when the elongation of initiated replicons or the joining of intermediate-sized DNA fragments is retarded during replication. They are consistent with the notions that H1 is lost from initiated replicons and/or the loss of H1 is part of an alarm response in the cell which might facilitate events leading to gene amplification.

Changes in nucleosome repeat lengths precede replication in the early replicating metallothionein II gene region of cells synchronized in early S phase.

Previous investigations showed that inhibition of DNA synthesis by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine produced large changes in the composition and nucleosome repeat lengths of bulk chromatin. Here we report results of investigations to determine whether the changes in nucleosome repeat lengths might be localized in the initiated replicons, as postulated [D'Anna, J. A., & Prentice, D. A. (1983) Biochemistry 22, 5631-5640]. In most experiments, Chinese hamster (line CHO) cells were synchronized in G1, or they were synchronized in early S phase by allowing G1 cells to enter S phase in medium containing 1 mM hydroxyurea or 5 micrograms mL-1 aphidicolin, a procedure believed to produce an accumulation of initiated replicons that arise from normally early replicating DNA. Measurements of nucleosome repeat lengths of bulk chromatin, the early replicating unexpressed metallothionein II (MTII) gene region, and a later replicating repeated sequence indicate that the changes in repeat lengths occur preferentially in the early replicating MTII gene region as G1 cells enter and become synchronized in early S phase. During that time, the MTII gene region is not replicated nor is there any evidence for induction of MTII messenger RNA. Thus, the results are consistent with the hypothesis that changes in chromatin structure occur preferentially in the early replicating (presumably initiated) replicons at initiation or that changes in chromatin structure can precede replication during inhibition of DNA synthesis. The shortened repeat lengths that precede MTII replication are, potentially, reversible, because they become elongated when the synchronized early S-phase cells are released to resume cell cycle progression.

Time-dependent changes in H1 content, H1 turnover, DNA elongation, and the survival of cells blocked in early S phase by hydroxyurea, aphidicolin, or 5-fluorodeoxyuridine.

Cells were synchronized in G1 by isoleucine deprivation and then released into medium containing 1 mM hydroxyurea (HU), 5 micrograms mL-1 aphidicolin (APC), or 1 microgram mL-1 5-fluorodeoxyuridine (fl5dU). Coulter volume, content of histone H1 per unit DNA, turnover of histone H1, the extent of DNA elongation, and the survival of cells were measured as functions of time after release into the presence of the drugs. At the concentrations used in the experiments, the drug differ in their toxicity (fl5dU greater than HU greater than APC), induction of unbalanced cell growth, and the distribution of new DNA fragment sizes allowed during block, but they all (1) allow cells to enter S phase, (2) cause similar time-dependent losses of histone H1 per unit DNA, which begin as synchronized G1 cells begin to enter S phase, (3) retard DNA elongation beyond replicon size, and (4) retard the turnover of histone H1. The results indicate that loss of histone H1, inhibition of histone turnover, the retarded ligation of newly replicated DNA into bulk chromatin, and chromatin structural changes may be part of the cell's general response to inhibition of DNA replication. Since transient S phase block increases the frequencies of gene amplification [Mariani, B. D., & Schimke, R. T. (1984) J. Biol. Chem. 259, 1901-1910] and sister chromatid exchanges (SCE) [Rainaldi, G., Sessa, M. R., & Mariani, T. (1984) Chromosoma 90, 46-49], the observed changes in H1 content and chromatin organization may also be essential features of gene amplification and SCE.

Extent of histone modifications and H1(0) content during cell cycle progression in the presence of butyrate.

The effects of butyrate upon the extents of phosphorylation of histones H1 and H1(0) during cell-cycle progression have been investigated. Chinese hamster (line CHO) cells were synchronized in early S phase and released into medium containing 0 or 15 mM butyrate to resume cell-cycle traverse into G1 of the next cell cycle. Cells were also mechanically selected from monolayer cultures grown in the presence of colcemid and 0 or 15 mM butyrate to obtain greater than 98% pure populations of metaphase cells. Although cell cycle progression is altered by butyrate, electrophoretic patterns of histones H1, H1(0), H3, and H4 indicate that butyrate has little, if any, effect on the extents of H1 and H1(0) phosphorylation during the cell cycle or the mitotic-specific phosphorylation of histone H3. Butyrate does, however, inhibit removal of extraordinary levels of histone H4 acetylation (hyperacetylation) during metaphase, and it appears to cause an increase in the content of H1(0) in chromatin during the S or G2 phases of the cell cycle.

G1- and S-phase syntheses of histones H1 and H1o in mitotically selected CHO cells: utilization of high-performance liquid chromatography.

We have employed high-performance liquid chromatography (HPLC) to investigate the syntheses of histones H1 and H1o as synchronized cells traverse from mitosis to S phase. Chinese hamster (line CHO) cells were synchronized by mitotic selection, and, at appropriate times, they were pulse labeled for 1 h with [3H]lysine. Histones H1 and H1o were extracted by blending radiolabeled and carrier cells directly in 0.83 M HC1O4; the total HC1O4-soluble, Cl3CCO2H-precipitable proteins were then separated by a modification of an HPLC system employing three mu Bondapak reversed-phase columns [Gurley, L. R., D'Anna, J. A., Blumenfeld, M., Valdez, J. G., Sebring, R. J., Donahue, D. K., Prentice, D. A., & Spall, W. D. (1984) J. Chromatogr. 297, 147-165]. These procedures (1) produce minimally perturbed populations of synchronized proliferating cells and (2) maximize the recovery of radiolabeled histones during isolation and analysis. Measurements of rates of synthesis indicate that the rate of H1 synthesis increases (3.6 +/- 0.5)-fold as cells traverse from early to mid G1; as cells enter S phase, the rate of H1 synthesis increases an additional congruent to 22-fold and is proportional to the number of S-phase cells. In contrast to H1, the rate of H1o synthesis is nearly constant throughout G1. As cells progress into S phase, the rate of H1o synthesis increases (3.1 +/- 0.2)-fold so that it also appears to be proportional to the number of S-phase cells. Except for the first 1-2 h after mitotic selection, these results are similar to those obtained when cells are synchronized in G1 with the isoleucine deprivation procedure.

Dephosphorylation of histones H1 and H3 during the isolation of metaphase chromosomes.

Histones have been extracted from isolated metaphase chromosomes prepared by the method of Wray and Sutbblefield [Exp. Cell Res 59, 469-478 (1970)] and by a Nonidet P-40 detergent procedure based on the method of Wigler and Axel [Nucleic Acids Res. 3, 1463-1471 (1976)]. Analysis of the densitometer profiles of long polyacrylamide gels shows that the mitotic phosphorylations of histone H1 (H1M) and histone H3 are extensively depleted during chromosome isolation. These data indicate that CHO metaphase chromosomes prepared by standard methodologies do not represent in vivo chromosomes with respect to their histone phosphorylations; therefore, current chemical and structural studies of isolated metaphase chromosomes may require further clarification.

Disruption of the proximal anastomosis of axillobifemoral grafts: two case reports.

The use of axillobifemoral grafting in the treatment of infected aortic prostheses or in the treatment of patients at high risk for aortoiliac occlusive disease has become a widely accepted treatment modality. Few complications involving the proximal anastomosis have been reported. The present report describes two patients who suffered proximal anastomotic dehiscence several weeks after surgery when they fully extended and raised the affected arm.

DNA contents of replication without DNA density labeling.

A new method for determining the timing of DNA replication in specific regions of the mammalian genome without the use of DNA density labeling and DNA density centrifugation is described. The method is based on determination of average relative DNA copy numbers in specific genomic regions as cells progress through S phase, and "time of replication" for a specific region is described in terms of the cell's DNA content when the region is replicated. DNA is isolated from synchronized populations of G1 and S phase cells, it is slot-blotted at the same DNA concentration(s) for each population, and it is hybridized with 32P-labeled DNA probes that are specific to the regions of interest. Quantitation of the slot blot autoradiograms and flow cytometric analysis allows determination of (a) average relative DNA copy numbers for the regions of interest in synchronized cell populations, and (b) the average total DNA content in each population of synchronized cells. This information and the flow cytometry histograms are then used to calculate the cellular DNA content at which each region of interest is replicated. The results have a precision of less than or equal to +/- 10% of S phase for Chinese hamster (line CHO) rhodopsin, metallothionein II, the 5'-end of dihydrofolate reductase, the telomeric repeated sequence, pHuR-093 (also located near the centromeres in CHO chromosomes), and the c-Ki-ras family.