Effects of protein kinase inhibitors 1(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and N-[2-guanidinoethyl]-5-isoquinolinesulfonamide hydrochloride (HA1004) on calcitriol-induced differentiation of HL-60 cells

HL-60 promyelocytic leukemia cells were induced to differentiate by 1,25-dihydroxyvitamin D3 (calcitriol) into mature monocytes. Differentiation was assessed by nitro blue tetrazolium dye reduction, nonspecific esterase activity, and DNA synthesis. Terminal differentiation of cultures induced by calcitriol (10 nM) was inhibited by 80% when cells were treated simultaneously with protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) (32 [mu]M) and N-[2-guanidinoethyl]-5-isoquionlinesulfonamide hydrochloride (HA1004) (320 [mu]M). The 50 for inhibition of calcitriol-induced differentiation was approximately 15 [mu]M for H-7 and 170 [mu]M for HA1004. The IC50 values for H-7 and HA1004 antagonism of calcitriol-induced differentiation are quantitatively and relatively correlated to their known action to inhibit protein kinase C activity. Treatment of cells with concentrations of 0-32 [mu]M H-7 or 0-320 [mu]M HA1004 alone did not affect cell growth, differentiation, or trypan blue exclusion. However, higher concentrations of H7 (> 32 [mu]M) and HA1004 (> 320 [mu]M) were found to be cytotoxic. The data presented suggest that calcitriol-induced differentiation is antagonized by inhibitors of protein kinase and are consistent with the hypothesis that kinase C activity is required for HL-60 cell differentiation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27400/1/0000432.pd

Area under the curve of methotrexate and creatinine clearance are outcome-determining factors in primary CNS lymphomas

Although high-dose methotrexate (HD-MTX) is the most effective drug against primary CNS lymphomas (PCNSL), outcome-determining variables related to its administration schedule have not been defined. The impact on toxicity and outcome of the area under the curve (AUC(MTX)), dose intensity (DI(MTX)) and infusion rate (IR(MTX)) of MTX and plasmatic creatinine clearance (CL(crea)) was investigated in a retrospective series of 45 PCNSL patients treated with three different HD-MTX-based combinations. Anticonvulsants were administered in 31 pts (69%). Age >60 years, anticonvulsant therapy, slow IR(MTX) (1100 micromol hl(-1) were independently associated with a better survival. Slow CL(crea) and high AUC(MTX) are favourable outcome-determining factors in PCNSL, while slow CL(crea) is significantly related to higher toxicity. AUC(MTX) significantly correlates with age, anticonvulsant therapy, IR(MTX), and DI(MTX). These findings, which seem to support the choice of an MTX dose >/=3 gm(-2) in a 4-6-h infusion, every 3-4 weeks, deserve to be assessed prospectively in future trials. MTX dose adjustments in patients with fast CL(crea) should be investigated

Materials and Methods

A modified gas-liquid chromatographic method for de-termining plasma concentrations of bupivacaine and lid-ocaine is described, with cyclizine as an internal standard. The extraction procedure requires no solvent evaporation, thus overcoming the problem of drug volatility. Concen-trations as low as 0.1 mg/liter can be determined. The plasma sample is made alkaline and extracted into n-hexane, re-extracted into a small volume of an aqueous acid phase, and finally extracted into 50 p1 of methylene chloride after alkallnization. The final extract is assayed by gas chromatography on a 5 % OV-17 column. The ex-traction scheme of the present method eliminates inter-ferences by endogenous plasma constituents. Bupivacaine (Marcaine) is a local anesthetic of the anilide type with an action longer than that of lidocaine (Xylocaine) and mepivacaine (Carbocaine) (1-3). Most of the systemic toxic reactions from the local anesthetics are correlated with high concentrations of these drugs in plasma, the peak values for bupivacaine or lidocaine after different regional anesthetic techniques ranging from 1.5 to 4.5 mg/liter. Signs of toxicity occur when the concentration in plasma exceeds 5 mg/liter (4). Determinations of these drugs in plasma can therefore make their use safer, and may help throw light on various phar-macokinetic processes occurring in patients. This information is of value to the clinician for the choice and the dosage of the drug to be used. Several gas-chromatographic methods for determination of bupivacaine (5-8) and lidocaine (9-12) have been reported. However, they involve various extraction procedures and the organic phase must be purified before the solvent is evapo-rated. These time-consuming procedures can lead to lower recovery and sensitivity due to adsorption of the drug to the glasswareand evaporation of volatile components. The method described herein allows more rapid measure-ment of bupivacaine and or lidocaine in plasma

Differential expression of cytosolic activation factors for NADPH oxidase in HL-60 leukemic cells

Activation of NADPH oxidase in undifferentiated HL-60 leukemic cells and in HL-60 cells differentiated along the myeloid pathway with dibutyryl cyclic AMP (dbcAMP) or dimethyl sulfoxide (Me2SO) was studied. Upon stimulation with a calcium ionophore, a phorbol ester, arachidonic acid or gamma-hexachlorocyclohexane, Me2SO-differentiated HL-60 cells generated superoxide (O2-) at higher rates than dbcAMP-differentiated cells. Undifferentiated cells generated O2- only at low rates upon stimulation with the above agents. In cell-free systems, NADPH oxidase activity was reconstituted by combining membranes of undifferentiated or dbcAMP- or Me2SO-differentiated HL-60 cells, cytosol of Me2SO-differentiated cells and arachidonic acid. This basal O2- formation was enhanced several-fold by guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]), a potent activator of guanine nucleotide-binding proteins. In contrast, cytosol of dbcAMP-differentiated cells reconstituted O2- formation only in the presence of GTP[gamma S], and cytosol of undifferentiated cells was inactive. Submaximally stimulatory amounts of cytosolic protein of Me2SO- and dbcAMP-differentiated cells synergistically stimulated O2- formation in the presence but not in the absence of GTP[gamma S]. We conclude that differentiations of HL-60 cells with Me2SO and dbcAMP are not equivalent with respect to activation of NADPH oxidase and that two cytosolic activation factors are involved in the regulation of this effector system