Computerised morphometrical analysis in endometrial hyperplasia for the prediction of cancer development. A long-term retrospective study from northern Norway.

AIMS: To evaluate and compare the long term prognostic value of the WHO classification and the computerised multivariate morphometrical D score in endometrial hyperplasia. To test the reproducibility of the D score in two different centres. METHODS: Histopathological WHO classification and computerised morphometrical analysis using the D score (< 0, high risk; > 1, low risk; 0-1, uncertain) in a population based study from northern Norway of archival dilatation and curettage material from 68 women with 10-20 years of follow up. RESULTS: Of the 68 patients included in the study, 18 developed cancer. The sensitivity and specificity of the D score (< 0 v > 1) were 100% and 78%, respectively, which was better than the WHO classification (89% and 60%, respectively). The negative and positive predictive values for the D score were 100% and 58% and of the WHO classification 94% and 44%, respectively. This study found a slightly higher specificity for the D score than former retrospective studies, but otherwise the results were comparable. The D score results were reproducible between the two centres (R = 0.91; slope = 0.98; intercept = 0.3). CONCLUSIONS: D score assessment is a reproducible and more accurate predictor of outcome of endometrial hyperplasia than the WHO classification assessed by an experienced gynaecological pathologist. Routine application of the D score might reduce over and undertreatment of endometrial hyperplasia.

A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F-actin and DNA.

Drug-initiated apoptosis of human leukemia HL-60, THP-1, and U-937 cells was studied via multiparameter flow cytometry and cell sorting. A new flow cytometric method that allows both identification and quantitation of apoptotic cells and estimation of their cell cycle specificity is presented. The method is based on paraformaldehyde fixation followed by staining of F-actin and DNA with fluorescein isothiocyanate (FITC)-phalloidin and propidium iodide (PI), respectively. Bivariate green fluorescence (F-actin) vs. side scatterplots of HL-60 cells treated with 10 microM etoposide for 4 h showed two cell populations, one with high green fluorescence and low side scatter and one with low green fluorescence and high side scatter. Sorting revealed cells with intact nuclei in the high green fluorescence/low side scatter population and cells with fragmented nuclei in the low green fluorescence/high side scatter population, demonstrating that the cells in the latter population were apoptotic. Exposure of HL-60 cells to 10 microM etoposide for 4 h resulted in S-phase selective apoptosis, whereas 5 micrograms/ml cycloheximide initiated apoptosis mainly in G0/G1-phase and S-phase cells. The apoptotic response of HL-60 cells to 20 GY gamma-irradiation was selective for S-phase and G2 + M-phase cells. The present method offers the opportunity to estimate the cell cycle distributions of both the apoptotic and the nonapoptotic cell populations, which is especially valuable when apoptosis occurs in association with cell cycle perturbations. A similar shift from one to two cell populations in green fluorescence vs. side scatter-plots, similar to that observed for HL-60 cells, was observed in the THP-1 and U-937 cell lines secondary to etoposide treatment.

Homocysteine increases the relative number of apoptotic cells and reduces the relative number of apoptotic bodies in HL-60 cells treated with 3-deazaadenosine.

The effect of exogenous homocysteine thiolactone (Hcy) on apoptosis initiated by 3-deazaadenosine (c3Ado) was studied in human leukemia HL-60 cells. Flow cytometric analysis allowed evaluation of the relative number of apoptotic cells (APC) to apoptotic bodies (APB) with a sub G0/G1 DNA content. Addition of 1 mM Hcy to HL-60 cells exposed to 5 to 100 microM c3Ado was followed by a large increase in the number of APC and a simultaneous decrease in the number of APB. The effects of Hcy on both the formation of APC and APB was dose-dependent; however, Hcy concentrations above 250 microM were required for inhibition of APB formation to take place. Fluorescence microscopic examination of unfixed cells stained with acridine orange demonstrated different morphology of APC between cultures treated with c3Ado or c3Ado plus Hcy. Whereas APC in cultures treated with 100 microM c3Ado displayed pronounced cytoplasmic membrane blebbing, only minor blebbing was displayed by APC in cultures treated with 25 microM c3Ado and 1 mM Hcy. Extensive nuclear fragmentation was observed in APC regardless of Hcy addition. By cell sorting we demonstrate the presence of APC with the same DNA content as viable G0/G1 and S-phase cells in cultures treated with 25 microM c3Ado and 1 mM Hcy, indicating that cells in all cell cycle phases undergo apoptosis in these cultures. Neither the formation of APC nor APB in apoptosis initiated by cycloheximide or dactinomycin were influenced by 1 mM Hcy. The Hcy effects on c3Ado apoptosis were abrogated in part by 3-deaza-(+/-)-aristeromycin, a more specific and potent inhibitor of S-adenosylhomocysteine hydrolase.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevation of cyclic AMP levels in HL-60 cells accumulated in G1 or G2 by transmethylation inhibitors.

Effects of the transmethylation inhibitors 3-deazaadenosine (c3Ado) and 3-deaza-(+/-)-aristeromycin (c3Ari) on cell cycle and cyclic AMP (cAMP) concentrations in human promyelocytic leukemia cells (HL-60) were studied by flow cytometry and radioimmunoassay techniques. Previously described cell cycle accumulations, after incubation with drugs (25 microM) for two cell doublings (36 hr), were localized to G1 and G2 after incubation with c3Ado and c3Ari, respectively. cAMP levels were elevated in cells treated with c3Ado (35%) and c3Ari (92%) for 36 hr. Addition of the phosphodiesterase (PDE) inhibitor theophylline, increased cAMP levels further, while cAMP responsiveness to the beta-adrenergic stimulator isoproterenol was attenuated after c3Ado and c3Ari incubation. Homocysteine thiolactone (Hcy) alone reduced cell growth slightly (5%) and increased cAMP levels (17%). Hcy increased the growth inhibitory effects of c3Ado, while no modulating effect was seen in combination with c3Ari, nor did Hcy counteract the effects on the cell cycle perturbations. The results suggest that c3Ado- and c3Ari-induced cell cycle accumulation is, at least in part, mediated through cAMP elevation, possibly due to PDE inhibition secondary to S-adenosyl-homocysteine hydrolase inhibition and S-adenosyl-homocysteine build-up.

Differential cell cycle perturbation by transmethylation inhibitors.

Cell cycle distribution of HL-60 cells was studied by flow cytometry after incubation with the transmethylation inhibitors 3-deaza-(+/-)-aristeromycin (c3 Ari) and 3-deazaadenosine (c3 Ado). Cells were incubated with the drugs (25 microM) for two cell doublings in control cells (36 hr). The presence of c3 Ari caused a dose-dependent, reversible G2 + M arrest, whereas c3 Ado-treated cells accumulated in G0/G1. The G2 + M arrest was also found in NIH/3T3 cells incubated for 36 hr with 25 microM c3 Ari, but not in U937 and K562 cells. Possible mechanisms for the described effects of c3 Ari are discussed from the perspective that inhibition of S-adenosyl homocysteine hydrolase, and subsequent inhibition of transmethylation reactions, at present is the only known site of action of c3 Ari.

Catecholamine binding and concentrations in acute phase plasma after surgery.

The present study was undertaken to decide whether the bound fractions and/or total concentrations of catecholamines were determinative for the variability of biologically active concentrations in human plasma. The binding and concentrations of noradrenaline (NA) and adrenalin (Adr) were determined in acute phase plasma after major hip surgery in five subjects. The bound fractions before surgery were 23.0% and 18.4% for NA and Adr, respectively. The binding of catecholamines increased in the post-operative period. Five days after surgery the binding of NA and Adr was 30.9% and 24.0%, respectively. The surgical trauma induced an acute phase reaction in plasma with a decrease of albumin (HSA) concentrations whereas the concentrations of alpha-1 acid glycoprotein (AAG) increased. The catecholamine concentrations showed a considerable inter- and intraindividual variability. However, the present work shows that the variability of the biologically active catecholamine concentrations is mainly dependent on the total plasma concentrations and not the plasma protein binding.

High-dose metoclopramide and chlorpromazine in the treatment of cisplatin-induced emesis.

Twenty patients with lung cancer, treated with cisplatin and etoposide, were divided into two groups at random and given antiemetic therapy consisting of either high-dose metoclopramide (MCL) intravenously (8 mg/kg over 7 hours) and chlorpromazine (CPZ) (50 mg orally), or a reduced dose of MCL (6 mg/kg over 7 hours) and CPZ (50 mg orally). Serum MCL concentrations were monitored during the infusions. In the two groups, 33% and 38% vomited during and after the courses, and antiemetic control was achieved in 83% and 75% of the patients. There was no significant difference between the groups, and side effects were negligible. MCL concentrations exceeded 0.7 microgram/ml in all patients, with great inter-individual variation.

High-dose methotrexate therapy (6-8 g/m2) in childhood malignancies: clinical tolerability and pharmacokinetics.

Five children, ages 2.5 to 12 years (mean 6.2 years), with acute lymphoblastic leukemia or non-Hodgkin's lymphoma were given 22 courses of high-dose methotrexate (HD-MTX) therapy (6-8 g/m2/24 h). No serious clinical complications were encountered, but stomatitis occurred after three (14%) of the courses. First-phase elimination half-lives (t1/2(alpha)) of MTX and 7-hydroxy-methotrexate (7-OH-MTX) after 21 infusions were 2.7 +/- 0.4 h and 6.5 +/- 1.8 h (mean +/- SD). In one course (4.5%) there was delayed systemic MTX elimination, with first-phase elimination half-lives (t1/2(alpha] for MTX and 7-OH-MTX of 4.2 and 9.9 h, respectively, and second-phase elimination half-lives (t1/2(beta)) of 43 and 58 h. Significant decreases in white blood cell count, increases in serum creatinine, and increases in alanine aminotransferase and/or aspartate aminotransferase during the first 2-6 days were present in five (23%), three (14%), and six (27%) of the courses, respectively. The regimen was tolerated well by the children.

Induction of HL-60 cell differentiation by 3-deaza-(+/-)-aristeromycin, an inhibitor of S-adenosylhomocysteine hydrolase.

The metabolically stable inhibitor of S-adenosylhomocysteine hydrolase (AdoHcyase), 3-deaza-(+/-)-aristeromycin (dzAri) has recently been shown to induce differentiation in HL-60 cells. The present study was undertaken to characterize the cytostatic, cytotoxic, and differentiation inducing properties of dzAri in HL-60 cells and to investigate biochemical consequences of AdoHcyase inhibition. A dye exclusion test and a clonogenic assay were used to test cytotoxic and cytostatic properties. dzAri had reversible cytostatic effects on HL-60 cells at concentrations lower than 10 microM and partially reversible cytotoxic effects above 10 microM. The induction of differentiation was dependent upon concentration and time of exposure, with maximal effect after 6 days incubation with 5-10 microM dzAri. Washout experiments demonstrated that the cells were not committed to differentiation after 48 h of incubation with dzAri. The AdoHcyase of HL-60 cells was inhibited with a Ki of 20 nM. The concentration of S-adenosylhomocysteine increased dose dependently 48 h after incubation with 0.1-100 microM dzAri. The incorporation of [3H]methyl from [methyl-3H]methionine into 5-methylcytosine of DNA was reduced by 26% at 5 microM dzAri. The findings indicate that continuous presence of dzAri is necessary to induce differentiation and inhibit proliferation in HL-60 cells. The inhibition of AdoHcyase perturbs levels of transmethylation metabolites and the incorporation of [3H]methyl into 5-methyl-cytosine of DNA.

Methotrexate measurements in plasma: comparison of enzyme multiplied immunoassay technique, TDx fluorescence polarization immunoassay, and high pressure liquid chromatography.

One hundred nine patient plasma samples were examined for methotrexate (MTX) levels by enzyme multiplied immunoassay technique (EMIT), fluorescence polarization immunoassay (TDx), and high pressure liquid chromatography (HPLC). EMIT analysis was performed twice within a time span of 18 months. All three methods measure MTX with a high degree of specificity, sensitivity, and precision. There was no evidence of decay of MTX concentrations in samples stored at -20 degrees C for 1.5 years. EMIT, TDx, and HPLC are adequate methods for MTX quantification in the clinical laboratory.

Pharmacokinetics of methotrexate and 7-hydroxy-methotrexate after high-dose (33.6 g/m2) methotrexate therapy.

We have measured MTX and 7-OH-MTX in plasma and urine samples from a 9-year-old boy treated with six consecutive 24-h IV high-dose MTX courses (33.6 g/m2) after a relapse of ALL. The between-course pharmacokinetics of MTX and 7-OH-MTX were found to be highly reproducible. Both MTX and 7-OH-MTX elimination followed a biphasic curve, initial half-lives (t1/2(alpha] being 2.86 +/- 0.44 h and 5.14 +/- 0.46 h (mean +/- SD) and second-phase biological half-lives (t1/2(beta] being approximately 18 and 16 h, respectively. The apparent volume of distribution for MTX was 0.8 L/kg, whereas the corresponding value for 7-OH-MTX was threefold less. Since clearance of MTX was within the range reported for lower doses, the data suggest that MTX pharmacokinetics are not dose-dependent up to 33.6 g/m2.