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R Rowley

Publications and source records attributed to R Rowley.

At least 19 recordsLinked to original sources
PubMed1996

Effect of B-type cyclin over-expression on radiation-induced mitotic delay in the fission yeast.

Exposure to ionizing radiation temporarily blocks eukaryotic cell cycle progression at the G2/M boundary (G2 delay). The delay probably provides time for repair of DNA damage before chromosome segregation and is thus an active response, indicative of a checkpoint control function. Transition from G2 into mitosis is normally controlled by the activity of a cyclin-dependent kinase, cdc2 in the fission yeast (Schizosaccharomyces pombe). Genetic and cell kinetic evidence suggest that irradiation may impose mitotic delay by inactivation of the cdc2 product, p34cdc2. The activity of p34cdc2 in G2 is regulated by phosphorylation and association with a B-type cyclin, the product of the cdc13 gene, p56cdc13. Previous work does not support a major role for changes in phosphorylation of p34cdc2 in the induction of mitotic delay. Alternatively the kinase may be regulated by changes in the activity/availability of p56cdc13. We have therefore tested the effect of high level, episomal expression of the cdc13 gene on the induction of mitotic delay. No influence of this procedure on the duration of delay was detected, either in a wild-type cell cycle background, or the mutants wee1-50 and cdc2-3w, which show abnormal phosphorylation of p34cdc2.

Cell Survival↗
PubMed1992

The rad3+ gene of Schizosaccharomyces pombe is involved in multiple checkpoint functions and in DNA repair.

A number of important molecular checkpoints are believed to control the orderly progression of cell cycle events. We have found that the radiation-sensitive Schizosaccharomyces pombe mutant rad3-136 is deficient in two molecular checkpoint functions. Unlike wild-type cells, the mutant cells are unable to arrest in the G2 phase of the cell cycle after DNA damage by gamma-irradiation and are also incapable of maintaining the dependence of mitosis upon the completion of DNA synthesis. An S. pombe genomic clone that complements the UV sensitivity of the rad3-136 mutant completely restores the missing checkpoint functions. The rad3+ gene is also likely to play a role in DNA repair.

Benomyl↗
PubMed1992

The wee1 protein kinase is required for radiation-induced mitotic delay.

Cellular feedback or 'checkpoint' mechanisms maintain the order of completion of essential, cell-cycle related functions. In the budding yeast, for example, the RAD9 gene product is required to delay progression into mitosis in response to DNA damage. Similarly, in fission yeast, the cdc25 and cdc2 gene products influence the ability of cells to delay mitosis in response to the inhibition of DNA synthesis. Because these two checkpoint controls regulate the same event, mitosis, we observed the effect of gamma-irradiation on cell cycle progression in fission yeast, to test whether the two controls require the same cell-cycle regulatory elements. We show that gamma-radiation-induced mitotic delay requires functional wee1 protein kinase but does not seem to involve the cdc25 pathway. Mitotic delay in response to DNA damage is thus distinct from the delay induced by inhibition of DNA synthesis, which involves cdc25 but is not dependent on wee1.

Cell Cycle Proteins↗
PubMed1992

Checkpoint controls in Schizosaccharomyces pombe: rad1.

'Checkpoint' controls ensure that the events of the cell cycle are completed in an orderly fashion. For example, such controls delay mitosis until DNA synthesis and repair of radiation-induced DNA damage are complete. The rad series of radiosensitive fission yeast mutants was examined to identify strains deficient for the DNA damage-responsive checkpoint control. Five were identified. A characterization of one (rad1-1) and the wild-type is presented. The rad1-1 mutant does not arrest after irradiation, is sensitive to killing by radiation and is not arrested by hydroxyurea, and thus is also deficient for the DNA synthesis-responsive checkpoint control. The radiosensitivity of the rad1-1 mutant was greatly reduced when irradiated and maintained for 6 h in a non-dividing (density inhibited) state, demonstrating that rad1-1 is repair proficient and radiosensitive only through failure to delay. The checkpoint controls for which rad1 is required appear to regulate G2-M progression through the activity of cdc2, here implicated in this role by the coincidence of the radiation transition point and the cdc2 execution point.

Cell Cycle↗
PubMed1992

Radiation-induced mitotic delay: a genetic characterization in the fission yeast.

Radiation-induced mitotic delay is under investigation in the fission yeast, Schizosaccharomyces pombe. A large range of cell cycle- and radiation-sensitive mutants of this yeast is available to facilitate this effort. Through an examination of such mutants it has been shown that the X-ray transition point and the p34cdc2 execution point are coincident; wee1- strains are not delayed by irradiation; and the radiation-sensitive mutants rad1-1, rad3-136, rad9-192, and rad17-W are not delayed by radiation or by inhibitors of DNA synthesis, including hydroxyurea. A model is proposed: Damaged DNA generates a signal to delay mitosis which is carried by the products of the rad genes to activate the tyrosine kinase p110wee1. This in turn inactivates the serine/threonine kinase p34cdc2, thereby blocking entry to mitosis. Unreplicated DNA also initiates a signal to delay mitosis which is carried by these same rad genes but, as indicated in the literature, transmission to p34cdc2 does not require p110wee1. The delay-deficient rad mutants may possess some properties of tumor suppressor genes, with implications for mutagenesis and oncogenesis.

Animals↗
PubMed1992

Reduction of radiation-induced G2 arrest by caffeine.

A large number of studies have been undertaken in an attempt to define the mechanism by which caffeine (1,3,7-trimethylxanthine) reduces the duration of radiation-induced arrest of cells in the G2 phase of the cell cycle. These studies are summarized and those agents which mimic the action of caffeine are listed in the order of their potency. This ranking does not match any activities of these agents described previously, but provides a comparison for future studies, which might profitably include measurement of the ability of these agents to inhibit protein kinases.

Animals↗
PubMed1991

stf1: a new suppressor of the mitotic control gene, cdc25, in Schizosaccharomyces pombe.

A novel element in the mitotic control, stf1, has been identified genetically by its ability to rescue cdc25-22 as well as a gene disruption of cdc25. This is the first phenotypically non-wee mutation shown to do so. stf1-1 functions additively with cdc2-1w, cdc2-3w, or wee1-6 to rescue cdc25. The available data are consistent with the wild-type gene product operating either on the same pathway as cdc25 or to stimulate cdc2 by a pathway independent of cdc25 or wee1. The stf1 gene has been cloned and sequenced and encodes a putative protein of 50-65 kD, depending on whether a potential intron is present. It is a novel protein with no homology detected in the current data bases. When challenged with hydroxyurea, stf1-1 acts additively with cdc2-3w in rescuing cdc25 mutants and in allowing mitosis to occur without DNA synthesis. It does not appear to play a role in the nutritional sensing pathway nor in the pathway mediating radiation-induced G2 delay.

Alleles↗
PubMed1990

Repair of radiation-induced chromatid aberrations: relationship to G2 arrest in CHO cells.

The literature suggests that the function of radiation-induced G2 arrest is to allow repair of potentially lethal damage before cell-entry into, and damage expression in, mitosis. The nature of the damage repaired is not known, but chromosome aberrations have been considered. To examine this possibility in G2 cells, the rate of repair of chromatid aberrations in CHO cells progressing to or arrested in G2 was compared with the rate of repair of the damage which gives rise to G2 arrest. To measure aberration repair rates, exponentially growing CHO cells arrested in G2 with 1.5, 2.5 or 3.5 Gy of X-rays were released into mitosis by treatment with 5 mM caffeine immediately or 1, 2 or 3 h after irradiation. Aberration frequencies in these cells were then related to the caffeine-free (repair) interval. To measure the rate of repair of arrest-causing damage a split-dose procedure was used. The half-times for aberration repair were approximately 1 h for achromatic gaps and 1.5 h for breaks, intrachanges and interchanges. The half-time for arrest damage repair varied with radiation dose. This result suggests that chromatid aberrations are not a primary cause of radiation-induced G2 arrest.

Animals↗
PubMed1990

Response of murine bone marrow granulocyte-macrophage colony-forming units to hyperthermia in situ.

A detailed understanding of how bone marrow stem cell progenitors are affected by heat is prerequisite to predicting how whole-body or regional hyperthermia protocols may affect bone marrow function. This investigation reports the reproductive integrity of murine tibial bone marrow granulocyte-macrophage colony-forming units (CFU-GM) after in situ hyperthermia. Heat was applied by water bath immersion of the leg of male BALB/c mice anesthetized with 90 mg/kg pentobarbital given subcutaneously. Tibial and rectal temperatures were monitored in representative animals by microthermocouples (tip diameter approximately 100 microns). By approximately 3 min after immersion of the limb, marrow temperature was within 0.3 degree C of water bath temperature (O'Hara et al., Int. J. Hyperthermia 5, 589-601, 1989) and was within 0.1 degree C by 5 min after immersion. The CFU-GM were cultured in "lung-conditioned" McCoy's 5A medium supplemented with 15% fetal calf serum and 0.3% Bacto agar. In situ heating of tibial marrow to exposure temperatures of 42, 42.5, 43, 44, and 45 degrees C gave D0's (+/- 95% CI) of 91 +/- 44, 44 +/- 27, 27 +/- 2.2, 16 +/- 6, and 7 +/- 4 min, respectively. Heating to 41.5 degrees C for up to 180 min did not result in cytotoxicity. Development of thermotolerance after approximately 100 min of heating was apparent by the presence of a "resistant tail" of the 42 degrees C survival curve. A plot of D0 vs water bath temperature was bimodal with an inflection point at approximately 42.5 degrees C. The inactivation enthalpy for temperatures above 42.5 degrees C was 586 kJ/mol (140 kcal/mol) and for temperatures below 42.5 degrees C was estimated to be 1205 kJ/mol (288 kcal/mol). These results show that CFU-GM can be heated predictably in situ, can be inactivated with thermal exposures as low as 42 degrees C, and are capable of developing thermotolerance. These findings underscore the necessity to understand stem cell inactivation by hyperthermia in situ prior to widespread implementation of clinical hyperthermia protocols where bone marrow may be included in the treatment field.

Adaptation, Physiological↗
PubMed1989

Novobiocin, nalidixic acid, etoposide, and 4'-(9-acridinylamino)methanesulfon-m-anisidide effects on G2 and mitotic Chinese hamster ovary cell progression.

Exponentially growing Chinese hamster ovary cells, exposed to inhibitors of topoisomerase II (novobiocin, nalidixic acid, etoposide, and 4'-(9-acridinylamino)methanesulfon-m-anisidide were blocked in progression through G2. The manner of recovery from the novobiocin-induced block, following drug removal, indicated that the blockade was at and before a specific point in G2 (a transition point). The transition point for novobiocin and putative transition points for nalidixic acid and 4'-(9-acridinylamino)methanesulfon-m-anisidide were about 30 min before metaphase. The transition point for nalidixic acid varied with concentration from about 70 min before metaphase, at 1 microgram/ml, to 24 min before metaphase at 15 micrograms/ml and above. The novobiocin- and nalidixic acid-induced G2 block could not be accounted for by cytotoxicity or DNA damage (detected by neutral elution). The novobiocin-induced G2 block could not be attributed to gross RNA synthesis inhibition. Progress beyond metaphase was blocked by novobiocin but not by nalidixic acid, when cells were exposed to drug concentrations which inhibited G2 cell progression. It is suggested that the progression of Chinese hamster ovary cells into but not through mitosis may require topoisomerase II.

Amsacrine↗
PubMed1988

Radioiodinated rhodamine-123: preparation and preliminary evaluation as an agent for tumor scintigraphy.

Mitochondria specific dye, rhodamine-123 (Rh-123), has been labeled with radioactive iodine and tissue distribution of the radiolabeled product has been studied in mice. Transplantable KHT sarcoma and a spontaneous adenomammary carcinoma served as tumor models. The FAB mass spectra of iodinated Rh-123 indicated that mono and di-iodo products were formed which, as shown by other in vitro tests, were positively charged, and were heavily taken up by the mitochondria of living cells in culture. In animals, it was observed that, initially the radioactivity was taken up by all major organs from which it cleared rapidly including that from the KHT sarcoma; but not from the spontaneous adenomammary carcinoma. As a result, the spontaneous tumors retained much higher radioactivity than the equal weight of blood (x4.3) or muscle (x9.5), and were unequivocally detectable by external scintigraphy. The mitochondria selectivity and the specificity of the radioiodinated Rh-123 for the tumors of epithelial origin are exciting and warrant further studies of its use in diagnosis and therapy.

Adenocarcinoma↗
PubMed1988

The effect of modulators of radiation-induced G2 arrest on the repair of radiation-induced DNA damage detectable by neutral filter elution.

It has been suggested that radiation-induced G2-arrest is an extension of interphase to allow repair of DNA double-strand breaks (dsb) prior to mitosis. Cycloheximide blocks the repair of the lesions which result in radiation-induced G2 arrest. Caffeine and cordycepin reduce the duration of G2 arrest. All of these agents should therefore reduce the cell's ability to repair dsb. The influence of cycloheximide (50 micrograms/ml), caffeine (5 mM) and cordycepin (0.15 mM) on the repair of the damage detectable in DNA by neutral filter elution was determined. Chinese hamster ovary cells (CHO) were irradiated with X-ray doses of 20, 60 and 100 Gy then allowed to repair without drug treatment or in the presence of each drug for intervals up to 6 h. DNA damage repair proceeded in two phases. The fast component of the repair process (t1/2 approx. 7 min) was not modified by drug treatment; the slow component (t1/2 approx. 170 min) was unaffected by cycloheximide or cordycepin, but appeared to be inhibited by caffeine. It was concluded that: (a) the lesion which results in radiation-induced G2 arrest is not the lesion which is detectable by neutral filter elution, and (b) the influence of caffeine on dsb repair is specific to caffeine and is not mediated by a reduction in the duration of G2 arrest.

Animals↗
PubMed1988

Method for probing cells in radiation-induced G2 arrest: demonstration of potentially lethal damage repair.

Chinese hamster ovary cells were arrested in the G2 phase of the cell cycle by X-irradiation. When subsequently treated with 5 mM caffeine the arrested population progressed into mitosis as a synchronous cohort where it was harvested by mitotic cell selection. This procedure provides a means to isolate cell populations treated in G2, for the investigation of G2 arrest. Comparisons were made of the number of cells retrieved from G2 arrest with the number suffering arrest, as determined by flow cytometry and by matrix algebraic simulations of irradiated cell progression. The retrieved population was not significantly less than expected for doses up to 3.5 Gy, indicating that the retrieval process does not favour the isolation of any population subset below this dose. Cell populations retrieved from arrest at varying intervals (0-3 h) after irradiation (0-3.5 Gy) showed an increase in survival with increase in interval, consistent with repair of potentially lethal damage. The repair curves (surviving fraction vs time) were each described by a single exponential. G2 cells that were brought to mitosis without a period of arrest exhibited the same radiation response as cells irradiated in mitosis.

Animals↗
PubMed1988

Effect of poly(adenosinediphosphoribose) synthesis inhibitors and structurally related compounds on radiation-induced G2 arrest.

A variety of poly(adenosinediphosphoribose) p(ADPR) synthesis inhibitors, structurally related compounds with no inhibitory activity, and agents which reduce radiation-induced G2 arrest, were tested for the concentration dependence of their effect on (a) CHO cell progression to mitosis, (b) the duration of G2 arrest in X-irradiated CHO cells and (c) [14C]NAD incorporation in permeabilized CHO cells, as a measure of p(ADPR) synthetase activity. Caffeine and nicotinamide uptake by viable cells was also measured. The concentration dependencies for reduction of radiation-induced G2 arrest and for p(ADPR) synthesis inhibition were markedly disparate, although all of the active inhibitors of p(ADPR) synthesis did reduce the duration of radiation-induced G2 arrest to some extent. These data indicate that p(ADPR) synthesis is not a requirement for the induction of G2 arrest by ionizing radiation.

Aminobenzoates↗
PubMed1988

Cell cycle effect on the induction of DNA double-strand breaks by X rays.

Filter elution was used to compare X-ray-induced DNA single- and double-strand breaks in proliferating (P) and quiescent (Q) cells of the 66 and 67 mouse mammary tumor lines. There was no difference either between cell type or between growth states in the amount of single-strand breaks as defined by elution at pH 12.2. In contrast, Q cells appeared to sustain a much larger amount of double-strand break damage per Gray than P cells, when the damage was measured by elution at either pH 7.2 or pH 9.6. Experiments which combined centrifugal elutriation with pH 7.2 elution demonstrated that G1-P cells were similar to Q (greater than or equal to 95% G1) cells in the induction of elution-detectable double-strand breaks, while the S-phase enriched fractions sustained less damage than G1-P, Q, or asynchronous P populations. Studies in which P populations were pulse labeled with [14C]thymidine confirmed this finding. Mathematical analysis of the elution kinetics of irradiated P, Q, and S-phase cells supports a model in which the complex elution profiles observed for P cells could be explained as the sum of the one-component exponential elution profiles of G1- and S-phase subpopulations. Also, the correlation between damage measured by pH 7.2 elution and cell survival was tested by examining the dose response for stimulated 66 cells (St4), which like Q cells are greater than or equal to 95% in G1 but are more resistant to X-ray-induced cytotoxicity than are the 66 Q cells. However, the induction of double-strand breaks in St4 cells was identical to that in Q cells. Thus we conclude that there is not necessarily a correlation between the amount of elution-detectable X-ray-induced double-strand breaks and cell survival.

Animals↗
PubMed1985

Is G2-arrest an active cellular response to irradiation?

Protein synthesis is normally required for G2-cell progression and for recovery from radiation-induced G2-arrest. In the presence of 5 mM caffeine this requirement is alleviated, indicating that the mechanism responsible for G2 cell progression actually remains intact in irradiated or protein synthesis inhibitor-treated cells. It is suggested that both radiation and cycloheximide-induced G2-arrest are not, therefore, passive consequences of cellular defects, but are rather, active cellular responses to the state of cellular integrity, implying the existence of G2 cell progression controls.

Animals↗
PubMed1985

Cell cycle age dependence for radiation-induced G2 arrest: evidence for time-dependent repair.

Exponentially growing eucaryotic cells, irradiated in interphase, are delayed in progression to mitosis chiefly by arrest in G2. The sensitivity of Chinese hamster ovary cells to G2-arrest induction by X rays increases through the cell cycle, up to the X-ray transition point (TP) in G2. This age response can be explained by cell cycle age-dependent changes in susceptibility of the target(s) for G2 arrest and/or by changes in capability for postirradiation recovery from G2-arrest damage. Discrimination between sensitivity changes and repair phenomena is possible only if the level of G2-arrest-causing damage sustained by a cell at the time of irradiation and the level ultimately expressed as arrest can be determined. The ability of caffeine to ameliorate radiation-induced G2 arrest, while inhibiting repair of G2-arrest-causing damage makes such an analysis possible. CHO cell monolayers were irradiated (1.5 Gy), then exposed to 5 mM caffeine for periods of 0-10 hr. Cell progression was monitored by the mitotic cell selection procedure. In the presence of caffeine, progression of irradiated cells was relatively unperturbed, but on caffeine removal, G2 arrest was expressed. The duration of G2 arrest was independent of the length of the prior caffeine exposure and, since cells of all ages were ultimately examined, the duration of arrest was also independent of cell cycle age at the time of irradiation. This finding indicates that the target for G2-arrest induction is present throughout the cell cycle and that the level of G2-arrest damage incurred is initially constant for all cell cycle phases. The data are consistent with the existence of a time-dependent recovery mechanism to explain the age dependence for radiation induction of G2 arrest.

Animals↗