Biochemical interactions between methotrexate and 1-β-d-arabinofuranosylcytosine in hematopoietic cells of children: a Pediatric Oncology Group study

Summary Children with acute lymphocytic leukemia (ALL) in remission were treated with overlapping sequential infusions of methotrexate (MTX) and 1--d-arabinofuranosylcytosine (araC) as part of continuation therapy. The doses and the sequence were chosen to mimic conditions that produced greater than additive antineoplastic activity with these two drugs in preclinical studies. To assess the potential for the drug combination to exhibit greater than additive effect in vivo, we investigated several biochemical parameters that had been associated with synergism in vitro. Because the patients were in remission, the intracellular parameters could only be measured in cytologically normal hematopoietic cells. We observed that (1) the mean plasma concentrations of MTX and araC were above those required to obtain a greater than additive cytotoxicity with the two drugs in tissue culture; (2) MTX did not have a significant antipurine effect in bone marrow mononuclear cells; (3) the mean intracellular concentration of deoxycytidine triphosphate (dCTP) was significantly lower after treatment with the drug combination than after therapy with araC alone; and (4) the ratio of araC triphosphate (araCTP) to dCTP was 2.6 times higher after treatment with the combination than after araC alone. These results indicate that it is possible to achieve in patients the biochemical conditions associated with the greater than additive antineoplastic activity of MTX and araC in vitro.Abbreviations ALL acute lymphocytic leukemia - araC 1--d-arabinofuranosyluracil - araCTP araC triphosphate - araU 1--d-arabinofuranosyluracil - dNTPs deoxyribonucleoside triphosphates - MTX methotrexate - TCA trichloroacetic acid Supported in part by grants from the National Cancer Institute, National Institutes of Health (CA-38 053; CA-33572, CA-32278, CA-38 859, CA-29 691, and CA-30 969). Preliminary reports on the biochemical data were published by E. M. N., A. M. T., and D. P. inProc Am Assoc Cancer Res 24: 133 (1983) and those on the clinical data, by R. A K., E. M. N., D. P. R. B. R., M. B. H., Y. R., and A. I. F. inProc Am Soc Clin Oncol 3: 201 (1984)     

Characterization of the muscarinic receptor subtype(s) mediating contraction of the guinea-pig lung strip and inhibition of acetylcholine release in the guinea-pig trachea with the selective muscarinic receptor antagonist tripitramine

1. The muscarinic receptor subtypes mediating contraction of the guinea-pig lung strip and inhibition of the release of acetylcholine from cholinergic vagus nerve endings in the guinea-pig trachea in vitro have previously been characterized as M₂-like, i.e. having antagonist affinity profiles that are qualitatively similar but quantitatively dissimilar compared to cardiac M₂ receptors. The present study sought to establish definitely the identity of these receptor subtypes by using the selective muscarinic receptor antagonist, tripitramine. Guinea-pig atria and guinea-pig trachea (postjunctional contractile response) were included for reference.
2. It was found that tripitramine antagonized methacholine-induced contractions of the guinea-pig lung strip with a pK_B value of 8.76±0.05. Both the parallel shifts of the concentration-response curves and the slope of the Schild plot being not significantly different from unity (when antagonist preincubation was for 2 h) indicated the involvement of a single population of receptors in the contractile response. From the pK_B values obtained with tripitramine and a range of other selective muscarinic receptor antagonists (cf. Roffel et al., 1993), this single population of receptors can only be classified as M₂-like.
3. Tripitramine antagonized methacholine-induced negative chronotropic and inotropic responses in guinea-pig right and left atria with apparent pK_B values of 9.4–9.6. However, such values were only obtained when antagonist preincubation was relatively long and/or antagonist concentration relatively high (e.g. with 1 h at 100 or 300 nM but 3 h at 30 nM). It thus appears that low concentrations of tripitramine do not readily equilibrate with M₂ receptors in guinea-pig atria nor with M₂-like receptors in the guinea-pig lung strip.
4. Tripitramine increased electrical field stimulation-induced cholinergic twitch contractions in guinea-pig trachea in concentrations of 0.3–100 nM, by blocking prejunctional muscarinic inhibitory autoreceptors; with higher concentrations, twitch contractions were progressively diminished, as a result of blocking postjunctional M₃ receptors (apparent pK_B value 6.07±0.15). The pEC₂₀ value (−log concentration that increases twitch by 20% of maximum) was 8.29±0.08, which would suggest that M₄ receptors are involved in this response.
5. Oxotremorine-induced inhibition of the release of prelabelled [³H]-acetylcholine from guinea-pig trachea, under conditions where there is no auto-feedback, was blocked by tripitramine (2 h preincubation) with a pK_B value of 8.56±0.06. The slope of the corresponding Schild plot was not significantly different from unity, which together with the parallel shifts of the concentration-response curves indicated the involvement of a single muscarinic receptor subtype.
6. Since the pK_B value for tripitramine at prejunctional receptors in guinea-pig trachea is in between the affinities towards M₂ and M₄ receptors, correlation plots were constructed to compare the pK_B values obtained with tripitramine and a range of other selective muscarinic receptor antagonists (cf. Kilbinger et al., 1995) to reported affinities at M₁–M₄ receptors. This showed rather similar distribution patterns of the data points around the line of equality in the case of M₂ and M₄ receptor subtypes. However, the correlation coefficient was markedly better for M₂ (0.9667) than for M₄ (0.5976). Since recent evidence suggests that M₄ receptors are not expressed in cholinergic nerves from guinea-pig trachea, it is concluded that prejunctional muscarinic autoinhibitory receptors in this tissue exhibit an atypical M₂ type character, with a pharmacological profile distinct from cardiac M₂ receptors.

     

Indikatorensätze in der Gesundheitsberichterstattung Probleme der Kompatibilität zwischen Gesundheitsindikatoren der Gesundheitsministerkonferenz der Bundesrepublik Deutschland und der WHO

In connection with the strategy Health for All 2000 the WHO developed health targets and health indicators which are applicable to all states to measure progress in health situation. In federal countries health statistics will be set up on the basis of an indicator set, agreed on by the ministers of health care of all federal countries at the end of 1991. After having included North Rhine-Westphalia into WHO and EU projects, it became necessary to examine the compatibility of existing data sets. In conclusion, proposals for improving comparability of health indicators are submitted.     

The Steven Johnson syndrome. A case study

Steven Johnson's Syndrome is a serious systemic disorder in which there are vesicobullous lesions involving the skin and mucous membranes. It can result as an immune response to an antigen or as a drug reaction. Most often it is considered as an allergic reaction. It is a self-limiting condition which responds to immediate management or may result in fluid loss, sepsis and death.     

Quality of Life following Radical Surgical Treatment of Gastric Carcinoma

Quality of life (QOL) in patients with gastric cancer who underwent total gastrectomy has so far not been studied using the EORTC QLQ-C30 (Quality of Life Core Questionnaire of the European Organization for Research and Treatment of Cancer) as a standardized European QOL instrument. The aim of this study was to evaluate the effect of radical procedures such as extensive lymph node resection and combined resection of adjacent organs on patients' QOL. From 1992 to 1996, 152 patients underwent total gastrectomy. All patients alive on July 1, 1996 were included in the study (77/152). For assessing QOL, the EORTC QOL questionnaire QLQ-C30 version 2.0 and a validated gastric cancer module were sent home to the patients for self-completion. The response rate was 91%. It was possible to evaluate the questionnaires of 62 patients who had undergone resection with curative intent including 13 extended gastrectomies (21%). Of the 62 resections, 50 were combined with D2 lymphadenectomy (80.6%). The global health status was not negatively influenced by D2 lymphadenectomy and extended gastrectomy. Patients with splenectomy were more affected by treatment than patients without splenectomy. Radical gastrectomy combined with D2 lymphadenectomy is the treatment of choice for gastric cancer patients, concerning not just survival but QOL as well.     

Characterization of soluble platelet guanylyl cyclase with peptide antibodies

Summary Soluble guanylyl cyclase partially purified from bovine and human platelets was characterized with antibodies raised against synthetic peptides corresponding to different sequences of the ₁- and ₁-subunits of the bovine lung enzyme. On immunoblots, the platelet guanylyl cyclase was recognized by the four antisera used, with the exception of an antiserum against the C-terminus of the ₁-subunit which did not react with the human platelet but with the bovine platelet ₁-subunit. Furthermore the human platelet ₁-subunit exhibited a slightly lower molecular mass than the bovine protein. The C-terminal antibodies precipitated native platelet and lung guanylyl cyclase activity. In contrast an antibody against a peptide out of the putative catalytic domain, which is highly conserved between all guanylyl cyclases sequenced so far, did not precipitate native guanylyl cyclase, although it recognized both subunits on immunoblots, suggesting that the respective amino acid sequence is located in an inner site of the protein.Abbreviations GC_pep2 YGPEVWEDIKKEA (one letter code) - GC_pep3 SRKNTGTEETEQDEN - GC_pep5 VYKVETVGDKYMTVSGLP - GC_pep8 KKDVEEANANFLGKASGID - TBS-T Tris-buffered saline, containing 0.0501o Tween 20 Correspondence to E. Böhme at the above address     

Calcium-binding proteins S100A8 and S100A9 as novel diagnostic markers in human prostate cancer.

PURPOSE: S100 proteins comprise a family of calcium-modulated proteins that have recently been associated with epithelial tumors. We examined the expression of two members of this family, S100A8 and S100A9, together with the S100 receptor RAGE (receptor for advanced glycation end products) in human prostate adenocarcinomas and in prostatic intraepithelial neoplasia. EXPERIMENTAL DESIGN: Tissue specimens of 75 patients with organ-confined prostate cancer of different grades were analyzed by immunohistochemistry for expression of S100A8, S100A9, and RAGE. In addition, in situ hybridization of S100A8 and S100A9 was done for 20 cases. An ELISA was applied to determine serum concentrations of S100A9 in cancer patients compared with healthy controls or to patients with benign prostatic hyperplasia (BPH). RESULTS: S100A8, S100A9, and RAGE were up-regulated in prostatic intraepithelial neoplasia and preferentially in high-grade adenocarcinomas, whereas benign tissue was negative or showed weak expression of the proteins. There was a high degree of overlap of S100A8 and S100A9 expression patterns and of S100A8 or S100A9 and RAGE, respectively. Frequently, a gradient within the tumor tissue with an increased expression toward the invaded stroma of the prostate was observed. S100A9 serum levels were significantly elevated in cancer patients compared with BPH patients or healthy individuals. CONCLUSION: Our data suggest that enhanced expression of S100A8, S100A9, and RAGE is an early event in prostate tumorigenesis and may contribute to development and progression or extension of prostate carcinomas. Furthermore, S100A9 in serum may serve as useful marker to discriminate between prostate cancer and BPH.     

Characterization of N-glucuronidation of the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in human liver: importance of UDP-glucuronosyltransferase 1A4.

The nicotine-derived tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is one of the most potent and abundant procarcinogens found in tobacco and tobacco smoke and is considered to be a causative agent for several tobacco-related cancers. Glucuronidation of the major metabolite of NNK, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), has been implicated as an important mechanism for NNK detoxification. To characterize NNAL metabolism by N-glucuronidation in humans, high-pressure liquid chromatography was used to detect glucuronide conjugates of NNAL formed in human liver microsomes in vitro. In addition to peaks corresponding to the O-glucuronides of NNAL (NNAL-O-Gluc), a second series of peaks were observed in human liver microsomes that were identified by liquid chromatography-mass spectrometry to be NNAL N-glucuronides (NNAL-N-Gluc). Microsomes prepared from liver specimens from individual subjects (n = 42) exhibited substantial variability in the levels of NNAL-N-Gluc (49-fold variability) and NNAL-O-Gluc (49-fold variability) formed in vitro. This variability was likely not due to differences in tissue quality, as substantial variability (5-fold) was also observed in the ratio of NNAL-N-Gluc/NNAL-O-Gluc formation, with a mean ratio of 1.7 in the 42 specimens. Liver microsomes from smokers (n = 14) exhibited no significant difference in the levels of either NNAL-N-Gluc or NNAL-O-Gluc formation, or in the ratio of NNAL-N-Gluc/NNAL-O-Gluc formation, as compared with liver microsomes from never smokers (n = 28). Overexpressed UDP-glucuronosyltransferase (UGT) 1A4 exhibited significant levels of N-glucuronidating activity (V(max)/K(m) = 3.11 microl. min(-1). g(-1)) in vitro; no NNAL-N-glucuronide formation was detected for the 11 other overexpressed UGT enzymes tested in these studies. These results demonstrate the importance of N-glucuronidation in the metabolism of NNAL and the role of UGT1A4 in this pathway.