Population pharmacokinetic modelling of factor IX activity after administration of recombinant factor IX in patients with haemophilia B.

PURPOSE: The aim is to develop a pharmacokinetic model for factor IX activity (FIX) after BeneFIX (nonacog alfa, rFIX) administration and assess potential covariates using all available clinical data collected during development. METHODS: The data set for model development combined observations from eight studies. Postdose FIX observations were adjusted by subtracting predose FIX if these were above the lower limit of quantification (BLQ) and all BLQ observations were removed. A population pharmacokinetic model was then developed with 4936 observations from 201 patients. Two additional studies (385 observations from 72 patients) became available and were used to evaluate the model. RESULTS: A two-compartment model, parameterized for clearance (CL), volume of distribution of the central (V1) and peripheral (V2) compartments, and intercompartmental clearance (Q), with an effect of weight on all parameters was the final model. Weight was incorporated as a power function with exponent estimates close to conventional allometric scaling. Including interoccasion variability (IOV) on CL and V1 showed decreases in the objective function. Investigations of a full block omega matrix lead to the retention of a correlation between V2 and Q. Age was not a significant covariate with weight already included in the model. Observations in the studies used for evaluation were found to be higher than simulated values immediately after dosing, as well as a week after dosing. The differences may be due perhaps to differences in the patients enrolled in the evaluation studies (all were adults) as well as the sample collection time after dosing (longer after dosing in the evaluation studies). CONCLUSIONS: FIX is appropriately modelled as a two-compartment model after rFIX administration. When weight is included, no additional effect of age is observed. Longer times of observation after dosing may be helpful in refining the model.

Mitosis-specific phosphorylation of vimentin by protein kinase C coupled with reorganization of intracellular membranes.

Using two types of anti-phosphopeptide antibodies which specifically recognize vimentin phosphorylated by protein kinase C (PKC) at two distinct PKC sites, we found that PKC acted as a mitotic vimentin kinase. Temporal change of vimentin phosphorylation by PKC differed form changes by cdc2 kinase. The mitosis-specific vimentin phosphorylation by PKC was dramatically enhanced by treatment with a PKC activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), while no phosphorylation of vimentin by PKC was observed in interphase cells treated with TPA. By contrast, the disruption of subcellular compartmentalization of interphase cells led to vimentin phosphorylation by PKC. Cytoplasmic and nuclear membranes are fragmented and dispersed in the cytoplasm and some bind to vimentin during mitosis. Thus, targeting of activated PKC, coupled with the reorganization of intracellular membranes which contain phospholipids essential for activation, leads to the mitosis-specific phosphorylation of vimentin. We propose that during mitosis, PKC may phosphorylate an additional subset of proteins not phosphorylated in interphase.

Severe hypoglycemia in a patient with anorexia nervosa.

OBJECTIVE: Severe hypoglycemia is an important but uncommon complication of anorexia nervosa. A 35-year-old woman showed severe hypoglycemia after a recovery from severe liver dysfunction due to malnutrition. METHODS: To reveal the cause of severe hypoglycemia, we measured plasma hormones and performed a 75g oral glucose loading test. RESULTS: Fasting serum adrenocorticotropic hormone, cortisol, growth hormone, somatostatin, and active ghrelin were elevated. Serum free triiodothyronine, leptin, and adiponectin were reduced. Plasma glucose fluctuated from 67 to 76 mg/dl after a 75g glucose ingestion, without hyperinsulinemia. Serum growth hormone, somatostatin, and active ghrelin levels were decreased after glucose ingestion. Plasma glucagon levels were increased and remained at high levels at 120 min, and glucose-dependent insulinotropic polypeptide (GIP) levels were continuously and remarkably increased after glucose ingestion. CONCLUSION: We observed a strongly reduced sensitivity in glucagon-induced hepatic glycogenolysis, and significantly elevated fasting and postprandial GIP levels, and a defective GIP-mediated glucagon secretion, in an anorectic patient with severe hypoglycemia.

Specific accumulation of Rho-associated kinase at the cleavage furrow during cytokinesis: cleavage furrow-specific phosphorylation of intermediate filaments.

The small GTPase Rho and one of its targets, Rho-associated kinase (Rho-kinase), are implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation and smooth muscle contraction. Since Rho also plays an essential role in cytokinesis, Rho-kinase may possibly mediate some biological aspects of cytokinesis. Here, using a series of monoclonal antibodies that can specifically recognize distinct phosphorylated sites on glial fibrillary acidic protein (GFAP) and vimentin, phosphorylation sites by Rho-kinase in vitro were revealed to be identical to in vivo phosphorylation sites on these intermediate filament (IF) proteins at the cleavage furrow in dividing cells. We then found, by preparing two types of anti-Rho-kinase antibodies, that Rho-kinase accumulated highly and circumferentially at the cleavage furrow in various cell lines. This subcellular distribution during cytokinesis was very similar to that of ezrin/radixin/moesin (ERM) proteins and Ser19-phosphorylated myosin light chain. These results raise the possibility that Rho-kinase might be involved in the formation of the contractile ring by modulating these F-actin-binding proteins during cytokinesis and in the phosphorylation and regulation of IF proteins at the cleavage furrow.

Glycosphingolipid patterns of rat kidney. Dependence on age and sex.

The composition and quantity of glycosphingolipids in kidney from 3-50-week-old male and female rats have been investigated. Glycosphingolipids were separated by high-performance liquid chromatography on a diethylaminoethyl-porous glass and porous glass column followed by high-performance thin-layer chromatography. The quantity of neutral glycolipids in male rat kidney decreased significantly between 3 and 25 weeks; the quantity in 25-week-old kidney became half the quantity found in 3-week-old kidney and remained constant thereafter. The quantity of neutral glycolipids in female rat kidney did not show age-dependent changes. The composition of neutral glycolipids revealed by high-performance thin-layer chromatography was constant, regardless of age and sex. Monoglycosylceramide and tetraglycosylceramide were the major compoments at all ages in both sexes. In contrast to the patterns of neutral glycolipids, ganglioside content was higher in female rats than males, and total ganglioside content increased in both sexes with increasing age. The most remarkable change was an increase of disialoganglioside in female rat kidney between 5 and 25 weeks. The ratio of disialogangliosides to monosialogangliosides (D/M value) in female rat kidney increased 4-fold during this period. The ganglioside pattern in female rat kidney was affected by spaying. The quantity of disialogangliosides decreased rapidly on the 7th day after spaying, but the level went back to normal on the 14th day after spaying. The data strongly suggest that sex hormones influence glycosphingolipid synthesis in rat kidney.

A case of pituitary adenoma with possible simultaneous secretion of thyrotropin and follicle-stimulating hormone.

A TSH- and FSH-screening pituitary adenoma was demonstrated in a 23-yr-old man who presented with bitemporal hemianopsia, but without clinical or laboratory evidences of hyper- or hypothyroidism or hypogonadism. Basal TSH (29-45 microunits/ml) and FSH (44-63 mIU/ml) were moderately elevated. GH and ACTH secretion were impaired, and basal PRL and testosterone were normal. TRH and LRH elicited a significant increase in TSH and FSH, respectively. The plasma glycoprotein alpha-subunit concentration was normal, and its response to simultaneous administration of TRH and LRH was low. Plasma TSH was not suppressed completely by exogenous T3. After operation and radiotherapy, elevated TSH and FSH as well as failure of T3 to suppress TSH were corrected. No significant changes in thyroid hormone concentration in the blood were detected in spite of the reduction of TSH. TSH and FSH concentrations in the tumor extract were lower than those in normal pituitaries. Histologically, the tumor was very vascular and consisted mostly of a single type of cell. The tumor cells were positively stained for FSH beta-subunit by immunohistochemical study, but for none of other pituitary hormones or subunits examined. Electron microscopic examination revealed relatively abundant single type secretory granules of high electron density. It is possible that the tumor simultaneously secreted TSH and FSH, the former possibly being immunologically active, but biologically inactive.

Comparison of the kinetic disposition and metabolism of E3810, a new proton pump inhibitor, and omeprazole in relation to S-mephenytoin 4'-hydroxylation status.

We studied the kinetic disposition and metabolism of E3810 [(+/-)-sodium 2-[[4-(3-methoxypropoxy)-3-methylpyridin-2-yl]methylsulfinyl ]-1H- benzimidazole], a new proton pump inhibitor, and omeprazole in 15 Japanese male volunteers, six of whom were poor metabolizers and nine of whom were extensive metabolizers of S-mephenytoin. All received once-daily 20 mg doses of E3810 or omeprazole for 7 days in a randomized crossover manner, with a 3-week washout period between the two trial phases. The parent drugs and their principal metabolites in plasma and urine were measured on days 1 and 7 after drug administration. The mean values for area under the plasma concentration-time curve (AUC) of omeprazole were 6.3- and 4.4-fold greater, whereas those of E3810 were 1.8- and 1.9-fold greater in poor metabolizers than in extensive metabolizers after the first and final doses, respectively. Although the mean AUC values for both drugs were significantly (p < 0.01 or p < 0.05) greater in poor metabolizers than in extensive metabolizers, the difference in the AUC between the two groups was smaller after E3810 than after omeprazole administration. The AUC of omeprazole tended to increase with the repeated doses in extensive metabolizers, whereas no such change was observed for E3810. The urinary excretions of the principal metabolite(s) of two proton pump inhibitors also reflected the data derived from plasma samples in relation to S-mephenytoin 4'-hydroxylation status. We conclude that the metabolism of two proton pump inhibitors is under coregulatory control of S-mephenytoin 4'-hydroxylase (CYP2C19), but that the magnitude of CYP2C19-mediated metabolism appears to differ between the two drugs. In contrast to omeprazole, the metabolism of E3810 is less saturable in extensive metabolizers during the repetitive dosings.

Comparison of the interaction potential of a new proton pump inhibitor, E3810, versus omeprazole with diazepam in extensive and poor metabolizers of S-mephenytoin 4'-hydroxylation.

OBJECTIVE: To compare the interaction potential of E3810, [(+/-)-sodium 2-[[4-(3-methoxpropoxy)-3-methylpyridin-2-yl]methylsulfinyl] -1H-benzimidazole] a new proton pump inhibitor, and omeprazole with diazepam in relation to S-mephenytoin 4'-hydroxylation status. STUDY DESIGN: Fifteen healthy male volunteers consisting of six poor metabolizers and nine extensive metabolizers of S-mephenytoin 4'-hydroxylation participated in the study, where two poor and three extensive metabolizers each as a group were randomly allocated to one of the three different treatment sequences with a 3-week washout period among the three trial phases. Each volunteer received an oral once-daily dose of E3810 (20 mg), omeprazole (20 mg), or placebo for 23 days and an intravenous dose (0.1 mg/kg) of diazepam on posttreatment day 8. Plasma concentrations of diazepam and demethyldiazepam were measured up to 16 days after the administration of diazepam. RESULTS: Diazepam was more slowly metabolized in the poor metabolizers than in the extensive metabolizers. No significant effects of E3810 and omeprazole on any kinetic parameters of diazepam were observed in the poor metabolizers. In the extensive metabolizers, omeprazole significantly decreased the mean clearance of diazepam and increased its half-life, area under the plasma concentration-time curve, and mean residence time compared with E3810 and placebo (p < 0.05 or 0.01), whereas no changes in these kinetic parameters were observed during the treatment with E3810. Omeprazole significantly increased the mean area under the plasma concentration-time curve (0-16 days) of demethyldiazepam in the extensive metabolizers compared with placebo (p < 0.01), whereas E3810 significantly increased it in the poor metabolizers compared with omeprazole or placebo (p < 0.05). CONCLUSION: The results indicate that E3810 as a substrate goes less toward S-mephenytoin 4'-hydroxylase (CYP2C19) and has a much weaker, if any, potential to interact with diazepam compared with omeprazole.

Acridones as inducers of HL-60 cell differentiation.

Fifteen acridone alkaloids were examined for their activity of induction of human promyelocytic leukemia cell (HL-60) differentiation. HL-60 cells were differentiated into mature monocyte/macrophage by atalaphyllidine (9), atalaphyllinine (12), and des-N-methylnoracronycine (13). The activities of NBT reduction, nonspecific esterase, and phagocytosis, were induced by 2.5 microM of 9, 12, and 13. After a 4-day treatment, 9, 12, and 13 at 10 microM inhibited clonal proliferation of HL-60 cells by 28, 96, and 63%, respectively. The structure-activity relationship established from the results revealed that hydroxyl group at C-1 and prenyl group at C-2 had an important role.

Expression of the angiogenic factor thymidine phosphorylase in human astrocytic tumors.

Thymidine phosphorylase (TP) has been implicated as a potent angiogenic factor and a prognostic factor in various human solid tumors. We investigated the expression of TP in a series of human astrocytic tumors using immunohistochemistry, enzyme-linked immunosorbent assay, and reverse transcriptase/polymerase chain reaction (RT-PCR) analysis. A total of 63 astrocytic tumors [27 glioblastomas (GBM), 19 anaplastic astrocytomas (AA), 17 low-grade astrocytomas (LGA)] and 5 normal brain tissues were immunohistochemically stained with antibodies to TP, vascular endothelial growth factor (VEGF), p53, MIB-1, and factor-VIII-related antigen. They were also evaluated for the degree of apoptosis by a ApopTag kit. Ten tumors (5 GBM, 2 AA, 3 LGA) and 3 normal brain tissues were evaluated for their expression of VEGF and TP by RT-PCR analysis. TP was constantly localized in the cytoplasm of astrocytic tumor cells, less intensely in the cytoplasm of vascular endothelial cells, but not in the normal brain. Some of the TP-positive cells were of macrophage origin, but most positive cells were the tumor cells themselves. Vascular density, MIB-1 positivity, p53 positivity, VEGF expression, and the apoptotic index were significantly higher in the TP-positive tumors than in TP-negative tumors. There was a significant correlation between TP and VEGF mRNA expression. In a limited number of glioblastoma cases, the apoptotic index was significantly higher in TP-positive glioblastomas than in TP-negative glioblastomas. In human astrocytic tumors, TP was expressed in the tumor, macrophage, and endothelial cells. TP was a potent angiogenic factor closely associated with cell proliferation and tumor apoptosis.

Comparison of the pharmacokinetics of E2020, a new compound for Alzheimer's disease, in healthy young and elderly subjects.

E2020, a central-acting cholinesterase inhibitor, is now under clinical development as a potential therapeutic agent for senile dementia of Alzheimer type. In the current study, the authors compared the pharmacokinetics of this drug after single oral administration in 12 healthy young volunteers (20-27 years of age) and 6 elderly volunteers (65-82 years of age). The subjects received a single 2-mg oral dose of E2020 after a meal. Blood samples for determination of the drug level were collected over 168 hours after drug administration and were measured by specific high-pressure liquid chromatography methods with ultraviolet detection. E2020 was generally well tolerated by all subjects of both groups. The plasma elimination half-life of the beta-phase (t 1/2 beta) and time to maximum peak plasma concentration (tmax) were significantly longer in the elderly than in the young: t 1/2 beta, 103.8 +/- 40.6 versus 59.7 +/- 16.1 hours; and tmax, 5.2 +/- 2.8 versus 3.4 +/- 1.5 hours, respectively. There were no statistically significant differences in maximum peak plasma concentration and area under the curve between the two groups. The mean (+/- standard deviation) oral clearance (Cl/F) in the elderly (9.1 +/- 2.4 L/h) was similar to that in the young (10.6 +/- 2.7 L/h). The volume of distribution in the steady state (Vdss/F) was significantly larger in the elderly than that in the young: 1217.2 +/- 223.2 versus 852.5 +/- 147.7 L, respectively. These results suggested that the drug was absorbed more slowly and distributed more widely and thoroughly, but that its clearance from the body is essentially unaffected by age.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase I study of E1077, a novel parenteral cephem antibiotic.

The safety and pharmacokinetics of E1077, a new injectable cephem antibiotic, were evaluated in healthy male adult volunteers. In the single-dose studies, 100, 250, 500, 1,000, and 2,000 mg of E1077 were administered by intravenous infusion at a constant rate for 60 minutes, then 1,000 mg of the drug by intravenous infusion at a constant rate for 5 minutes. The Cmax were 6.4, 15.7 +/- 12.0, 34.7 +/- 4.6, 63.2 +/- 4.6, 142.7 +/- 5.6, and 131.6 +/- 36.0 (means +/- SD) micrograms/mL, respectively, and the Cmax and AUC increased linearly with the dose. Plasma concentration-time curves were well described by a two-compartment open model. The plasma elimination half life of the drug was 1.88 +/- 0.15 hours. The mean urinary recovery within the first 24 hours was 94.1 +/- 5.1% of the dose. In the multiple-dose study, 2,000 mg of E1077 was intravenously administered at a constant rate over 60 minutes every 12 hours for 4.5 days (a total of nine times). The Cmax after the first and ninth doses were 134.0 +/- 17.4 and 135.5 +/- 15.5 micrograms/mL, respectively, and trough levels in day 1 and day 5 (at 12 hours after the first and ninth administration, respectively) were 2.2 +/- 0.8 and 1.9 +/- 0.4 micrograms/mL, respectively. No accumulation of the drug in plasma was observed. There were no significant differences in plasma levels or in the urinary recoveries between the single- and multiple-dose regimens.(ABSTRACT TRUNCATED AT 250 WORDS)

Phase I study of E1040, a new parenteral cephem antibiotic.

The safety and pharmacokinetics of E1040, a new injectable cephem antibiotic, were evaluated in healthy volunteers. In single-dose studies, 125, 250, 500, 1000 and 2000 mg of E1040 were administered by I.V. infusion over 1 hour. Results of 5 minutes I.V. infusions of 500, 1000 and 2000 mg of the drug were also studied. Plasma concentration-time profiles were well suited to a two-compartment open model. The half-life of elimination from plasma was 1.85 +/- 0.16 hours, and the Cmax and AUC paralleled the doses given. The mean urinary recovery within the first 24 hours was 85.7 +/- 6.43% of the dose. In a multiple-dose study, 2000 mg of E1040 (I.V. over 1 hour) was administered every 12 hours (total 9 times) and no abnormal accumulation of the drug in plasma was observed. There were no significant differences in plasma levels or in urinary recoveries between single- and multiple-dose regimens. There were no subjective or objective abnormal findings definitely attributable to the drug except that one subject given 250 mg over 1 hour reported diarrhea, and another complained of nausea during the infusion of 2000 mg over 5 minutes. From these results E1040 was concluded to be safe and well tolerated.

Functional partitioning of energy reserves by larvae of the marine bryozoan Bugula neritina (L.).

The effects of extended swimming on short-lived lecithotrophic larvae of the marine bryozoan Bugula neritina (L.) were examined. Larvae were forced to swim for 2 or 24 h by bath application of serotonin. Settlement and metamorphosis success were significantly reduced, larval dimensions were unaffected and ancestrulae were smaller after 24 h of swimming. Larvae settled predominantly on seawater-conditioned glass after 2 h, but became less discriminative after 24 h. Lipid content in intact larvae and dissociated surface ciliated and interior cell fractions was analysed by thin-layer chromatography. Hydrophilic lipids were unaffected by swimming regime. The hydrophobic fraction contained triglyceride, confirmed by proton nuclear magnetic resonance spectroscopy (1H-NMR) analysis and correlation spectroscopy (1H-1H COSY) patterns, which was significantly depleted after 24 h, and diacylglycerol, which was not. NMR spectra suggested no differences in fatty acid chain compositions between larvae swimming for 2 and 24 h. Triglyceride depletion was limited to the ciliated cell fraction. We propose that the functional partitioning of lipid reserves has evolved in association with the costs and benefits linked with larval dispersal.

Pituitary adenoma composed of two compartments each secreting prolactin or growth hormone.

We report a case of pituitary adenoma with two compartments, i.e. a part within the sella turcica and a part infiltrating the sphenoid bone beneath the sella, producing growth hormone (GH) and prolactin. The patient had the amenorrhea-galactorrhea syndrome, but not acromegalic symptoms. Although the two compartments were united through a small dural perforation, immunohistochemical studies demonstrated that GH and prolactin were separately secreted from the intrasellar and extrasellar components, respectively. Somatotropic adenomas frequently produce prolactin, but it is unusual for these hormones to be secreted from distinctly different parts of the same lesion.

Suprasellar ectopic pituitary adenoma: case report and review of the literature.

The occurrence of a totally suprasellar ectopic pituitary adenoma in a 71-year-old man is described. The tumor was attached to the pituitary stalk, extending upward toward the third ventricle. No intrasellar lesion was observed. Histological examination revealed a pituitary adenoma with large numbers of eosinophilic cells with moderate nuclear polymorphism and rare mitosis. Immunohistochemical staining revealed that the tumor cells were strongly positive for anti-adrenocorticotropic hormone antibody. A review of five previously reported intracranial ectopic pituitary adenomas revealed that two were silent corticotropic tumors and two occurred with Cushing's syndrome.

Estimation of growth fraction with bromodeoxyuridine in human central nervous system tumors.

Twenty-five patients with tumors of the central nervous system received bromodeoxyuridine (BUdR), 200 mg/sq m, by intravenous infusion every 8 hours for 3 days before surgery. Excised tumor specimens were fixed in chilled 70% ethanol, embedded in paraffin, and cut into 6-micron sections. Each section was reacted with monoclonal antibodies against BUdR and stained with immunoperoxidase to identify nuclei that had incorporated BUdR. The growth fraction of each tumor was estimated by calculating the ratio of BUdR-positive nuclei to the total number of tumor cells in three to six microscopic fields in viable areas of the tumor. In seven cases, the tumor doubling time was measured from the serial computerized tomography scans and an attempt was made to estimate the cell cycle time. The growth fractions ranged from 9.1% to 46.5% in malignant gliomas, 2.0% to 6.7% in low-grade gliomas, 11.2% to 43.2% in metastatic brain tumors, 0.8% to 1.9% in pituitary adenomas, 3.9% to 4.6% in acoustic neurinomas, and 6.2% to 8.2% in meningiomas and cerebellar hemangioblastomas. The estimated cell cycle time was 5 to 12 days in most malignant gliomas and brain metastases; however, the actual cell cycle time should be substantially shorter because cell loss was not considered in the calculation. Although the growth fraction appeared to correlate with the biological malignancy of each tumor, the tumor doubling time did not reflect growth potential. It is possible that unpredictable cell loss plays an important role in tumor growth at certain sizes. Therefore, the cell cycle times calculated in this study are considerably overestimated and should be interpreted with caution.

Effect of citrus flavonoids on HL-60 cell differentiation.

Twenty-seven Citrus flavonoids were examined for their activity of induction of terminal differentiation of human promyelocytic leukemia cells (HL-60) by nitro blue tetrazolium (NBT) reducing, nonspecific esterase, specific esterase, and phagocytic activities. 10 flavonoids were judged to be active (percentage of NBT reducing cells was more than 40% at a concentration of 40 microM), and the rank order of potency was natsudaidain, luteolin, tangeretin, quercetin, apigenin, 3, 3, '4, '5, 6, 7, 8-heptamethoxyflavone, nobiletin, acacetin, eriodictyol, and taxifolin. These flavonoids exerted their activity in a dose-dependent manner. HL-60 cells treated with these flavonoids differentiated into mature monocyte/macrophage. The structure-activity relationship established from comparison between flavones and flavanones revealed that ortho-catechol moiety in ring B and C2-C3 double bond had an important role for induction of differentiation of HL-60. In polymethoxylated flavones, hydroxyl group at C3 and methoxyl group at C8 enhanced the differentiation-inducing activity.

Effect of coumarins on HL-60 cell differentiation.

Twenty-eight coumarins, including 7 furocoumarins, were examined for their activity of induction of terminal differentiation of human promyelocytic leukemia cells (HL-60) by nitro blue tetrazolium (NBT) reducing, nonspecific esterase, specific esterase and phagocytic activities. Esculetin, nordalbergin, 6,7-dihydroxy-4-methylcoumarin and imperatorin had strong activity among the coumarins examined. HL-60 cells treated with these coumarins differentiated into mature monocyte/macrophage. The structure-activity relationship established from the results revealed that 6,7-dihydroxy moiety had an important role in the induction of differentiation of HL-60.

The antiproliferative effect of coumarins on several cancer cell lines.

Twenty-one coumarins were examined for their antiproliferative activity towards several cancer cell lines, namely lung carcinoma (A549), melanin pigment producing mouse melanoma (B16 melanoma 4A5), human T-cell leukemia (CCRF-HSB-2), and human gastric cancer, lymph node metastasized (TGBC11TKB). The structure-activity relationship established from the results revealed that the 6,7-dihydroxy moiety had an important role for their antiproliferative activity. Analysis of cell cycle distribution indicated that esculetin-treated cells accumulated in the G1 (at 400 microM) or in S phase (at 100 microM).