(1/1655) Caffeine can override the S-M checkpoint in fission yeast.
The replication checkpoint (or 'S-M checkpoint') control prevents progression into mitosis when DNA replication is incomplete. Caffeine has been known for some time to have the capacity to override the S-M checkpoint in animal cells. We show here that caffeine also disrupts the S-M checkpoint in the fission yeast Schizosaccharomyces pombe. By contrast, no comparable effects of caffeine on the S. pombe DNA damage checkpoint were seen. S. pombe cells arrested in early S phase and then exposed to caffeine lost viability rapidly as they attempted to enter mitosis, which was accompanied by tyrosine dephosphorylation of Cdc2. Despite this, the caffeine-induced loss of viability was not blocked in a temperature-sensitive cdc2 mutant incubated at the restrictive temperature, although catastrophic mitosis was prevented under these conditions. This suggests that, in addition to S-M checkpoint control, a caffeine-sensitive function may be important for maintenance of cell viability during S phase arrest. The lethality of a combination of caffeine with the DNA replication inhibitor hydroxyurea was suppressed by overexpression of Cds1 or Chk1, protein kinases previously implicated in S-M checkpoint control and recovery from S phase arrest. In addition, the same combination of drugs was specifically tolerated in cells overexpressing either of two novel S. pombe genes isolated in a cDNA library screen. These findings should allow further molecular investigation of the regulation of S phase arrest, and may provide a useful system with which to identify novel drugs that specifically abrogate the checkpoint control. (+info)
(2/1655) Increased sensitivity of hydroxyurea-resistant leukemic cells to gemcitabine.
Tumor cell resistance to certain chemotherapeutic agents may result in cross-resistance to related antineoplastic agents. To study cross-resistance among inhibitors of ribonucleotide reductase, we developed hydroxyurea-resistant (HU-R) CCRF-CEM cells. These cells were 6-fold more resistant to hydroxyurea than the parent hydroxyurea-sensitive (HU-S) cell line and displayed an increase in the mRNA and protein of the R2 subunit of ribonucleotide reductase. We examined whether HU-R cells were cross-resistant to gemcitabine, a drug that blocks cell proliferation by inhibiting ribonucleotide reductase and incorporating itself into DNA. Contrary to our expectation, HU-R cells had an increased sensitivity to gemcitabine. The IC50 of gemcitabine was 0.061 +/- 0.03 microM for HU-R cells versus 0.16 +/- 0.02 microM for HU-S cells (P = 0.005). The cellular uptake of [3H]gemcitabine and its incorporation into DNA were increased in HU-R cells. Over an 18-h incubation with radiolabeled gemcitabine (0.25 microM), gemcitabine uptake was 286 +/- 37.3 fmol/10(6) cells for HU-R cells and 128 +/- 8.8 fmol/10(6) cells for HU-S cells (P = 0.03). The incorporation of gemcitabine into DNA was 75 +/- 6.7 fmol/10(6) cells for HU-R cells versus 22 +/- 0.6 fmol/10(6) cells for HU-S cells (P < 0.02). Our studies suggest that the increased sensitivity of HU-R cells to gemcitabine results from increased drug uptake by these cells. This, in turn, favors the incorporation of gemcitabine into DNA, resulting in enhanced cytotoxicity. The increased sensitivity of malignant cells to gemcitabine after the development of hydroxyurea resistance may be relevant to the design of chemotherapeutic trials with these drugs. (+info)
(3/1655) RAD53 regulates DBF4 independently of checkpoint function in Saccharomyces cerevisiae.
The Cdc7p and Dbf4p proteins form an active kinase complex in Saccharomyces cerevisiae that is essential for the initiation of DNA replication. A genetic screen for mutations that are lethal in combination with cdc7-1 led to the isolation of seven lsd (lethal with seven defect) complementation groups. The lsd7 complementation group contained two temperature-sensitive dbf4 alleles. The lsd1 complementation group contained a new allele of RAD53, which was designated rad53-31. RAD53 encodes an essential protein kinase that is required for the activation of DNA damage and DNA replication checkpoint pathways, and that is implicated as a positive regulator of S phase. Unlike other RAD53 alleles, we demonstrate that the rad53-31 allele retains an intact checkpoint function. Thus, the checkpoint function and the DNA replication function of RAD53 can be functionally separated. The activation of DNA replication through RAD53 most likely occurs through DBF4. Two-hybrid analysis indicates that the Rad53p protein binds to Dbf4p. Furthermore, the steady-state level of DBF4 message and Dbf4p protein is reduced in several rad53 mutant strains, indicating that RAD53 positively regulates DBF4. These results suggest that two different functions of the cell cycle, initiation of DNA replication and the checkpoint function, can be coordinately regulated through the common intermediate RAD53. (+info)
(4/1655) Sustained induction of fetal hemoglobin by pulse butyrate therapy in sickle cell disease.
High levels of fetal hemoglobin (Hb F) protect from many of the complications of sickle cell disease and lead to improved survival. Butyrate and other short chain fatty acids were previously shown to increase Hb F production in erythroid cells in vitro and in animal models in vivo. However, butyrates are also known to inhibit the proliferation of many cell types, including erythroid cells. Experience with the use of butyrate in animal models and in early clinical trials demonstrated that the Hb F response may be lost after prolonged administration of high doses of butyrate. We hypothesized that this loss of response may be a result of the antiproliferative effects of butyrate. We designed a regimen consisting of intermittent or pulse therapy in which butyrate was administered for 4 days followed by 10 to 24 days with no drug exposure. This pulse regimen induced fetal globin gene expression in 9 of 11 patients. The mean Hb F in this group increased from 7.2% to 21.0% (P <.002) after intermittent butyrate therapy for a mean duration of 29.9 weeks. This was associated with a parallel increase in the number of F cells and F reticulocytes. The total hemoglobin levels also increased from a mean of 7.8 g/dL to a mean of 8.8 g/dL (P <.006). The increased levels of Hb F were sustained in all responders, including 1 patient who has been on pulse butyrate therapy for more than 28 months. This regimen, which resulted in a marked and sustained increase in Hb F levels in more than two thirds of the adult sickle cell patients enrolled in this study, was well tolerated without adverse side effects. These encouraging results require confirmation along with an appropriate evaluation of clinical outcomes in a larger number of patients with sickle cell disease. (+info)
(5/1655) Radiation therapy with concomitant hydroxyurea and fluorouracil in stage II and III head and neck cancer.
PURPOSE: In 1986, a multi-institutional phase II trial was begun to study the use of chemotherapy with concomitant radiation in patients with stage II and III head and neck cancer. End points were overall survival, progression-free survival, local/regional control, and toxicity in the setting of organ preservation with concomitant treatment. METHODS: Eligible patients with stage II or III disease received chemotherapy and radiation on a 2-week cycle. Chemotherapy consisted of continuous infusion fluorouracil (5-FU) at 800 mg/m2/d for 5 consecutive days (days 1 to 5) and hydroxyurea (HU) at 1 g orally every 12 hours for 13 doses starting the evening before the start of irradiation. Radiation therapy was given as single 1.8- to 2.0-Gy fractions for 5 consecutive days (days 1 to 5) with chemotherapy. Each 5 days of treatment was followed by a 9-day break (days 6 to 14), during which no additional treatment was given. Treatment cycles were repeated until the completion of the planned radiation dose (six to eight cycles). RESULTS: Between 1989 and 1996, 60 patients were enrolled. All patients were eligible for analysis, with a median follow-up of 52 months for surviving patients and 42 months for all patients. Grade 3 to 4 mucositis occurred in 57% of patients. The 5 year-actuarial overall survival, progression-free survival, and local/regional control were 65%, 82%, and 86%, respectively. Eight patients developed local and/or regional recurrence after treatment. Surgical salvage was possible in three of these patients. Thus, the ultimate 5-year local/ regional control was 91%. CONCLUSION: Concomitant radiation and chemotherapy with 5-FU and HU is an effective regimen in patients with stage II and III head and neck cancer. Progression-free survival and local/regional control appear to be superior to expected rates in patients treated with surgery and radiation. Further testing of this regimen in a phase III setting is indicated. (+info)
(6/1655) Cell cycle-dependent sequencing of cell fate decisions in Caenorhabditis elegans vulva precursor cells.
In Caenorhabditis elegans, the fates of the six multipotent vulva precursor cells (VPCs) are specified by extracellular signals. One VPC expresses the primary (1 degrees ) fate in response to a Ras-mediated inductive signal from the gonad. The two VPCs flanking the 1 degrees cell each express secondary (2 degrees ) fates in response to lin-12-mediated lateral signaling. The remaining three VPCs each adopt the non-vulval tertiary (3 degrees ) fate. Here I describe experiments examining how the selection of these vulval fates is affected by cell cycle arrest and cell cycle-restricted lin-12 activity. The results suggest that lin-12 participates in two developmental decisions separable by cell cycle phase: lin-12 must act prior to the end of VPC S phase to influence a 1 degrees versus 2 degrees cell fate choice, but must act after VPC S phase to influence a 3 degrees versus 2 degrees cell fate choice. Coupling developmental decisions to cell cycle transitions may provide a mechanism for prioritizing or ordering choices of cell fates for multipotential cells. (+info)
(7/1655) Randomized trial of zileuton in patients with moderate asthma: effect of reduced dosing frequency and amounts on pulmonary function and asthma symptoms. Zileuton Study Group.
This 6-month, randomized, multicenter study was designed to determine whether patients who had been treated with the leukotriene pathway inhibitor zileuton 600 mg four times daily (QID) for 2 months could be maintained at the same level of pulmonary function, symptom control, and beta-agonist use with less frequent dosing--first 600 or 800 mg three times daily (TID) and then twice daily (BID). A total of 278 patients with chronic asthma, ages 16 to 70, participated at 25 US centers. All had a 1-second forced expiratory volume (FEV1) of 35%-75%, reversible airway disease, and a nonsmoking history of 1 year. An 8-week open-label period (zileuton 600 mg QID) was followed by a 16-week double-blind period, in which patients who responded to the QID treatment were randomized to receive zileuton 600 or 800 mg TID for 8 weeks and then rerandomized to receive zileuton 600 or 800 mg BID for another 8 weeks. Primary outcomes were FEV1 and asthma symptom scores; secondary outcomes were peak expiratory flow rate, beta-agonist use, and asthma exacerbations requiring steroid rescue. Patients who showed improvements in lung function when treated with zileuton 600 mg QID demonstrated minimal decreases in FEV1 and comparable peak expiratory flow rates, symptom control, beta-agonist use, and systemic corticosteroid rescue when being treated with lower doses and/or less frequent doses of zileuton. Patients who demonstrate improved asthma control with zileuton 600 mg QID may be able to reduce their daily dosage and/or frequency while still maintaining the same level of symptom control. (+info)
(8/1655) Safety and clinical efficacy of zileuton in patients with chronic asthma.
Zileuton, a leukotriene pathway inhibitor used to treat asthma, improves lung function, relieves symptoms, and is well tolerated. The purpose of this 12-month, parallel-group, open-label study was to assess the efficacy of zileuton and evaluate liver function in patients treated with this drug (approximately 2% of patients treated with zileuton in controlled trials had reversible liver enzyme elevations). A total of 2,947 patients at 233 centers in the United States were randomly assigned in a 5:1 ratio to treatment with zileuton plus usual asthma care or usual asthma care alone. Efficacy variables included asthma exacerbations; need for alternative treatment, steroid rescue, emergency care, and hospitalizations; forced expiratory volume in 1 second (FEV1); and asthma symptom scores. The safety evaluation included measurement of alanine aminotransferase levels. Patients treated with zileuton had significantly fewer corticosteroid rescues (P < 0.001), required less emergency care (P < 0.05), had fewer hospitalizations, and had greater increases in FEV1 (P = 0.048). They also had significantly greater improvements in asthma symptoms. Increases in alanine aminotransferase levels to three times or more the upper limit of normal occurred in 4.6% of patients treated with zileuton and 1.1% of those receiving usual care (P < 0.001); most increases occurred during the first 2 to 3 months. Alanine aminotransferase levels decreased to less than two times the upper limit of normal or to baseline levels during zileuton treatment or after drug cessation. Jaundice or chronic liver disease did not develop in any patient. Adding zileuton to the therapeutic regimens of patients with asthma is likely to improve asthma control and lower utilization of healthcare resources. (+info)