Influence of cytokines onmdr1 expression in human colon carcinoma cell lines: Increased cytoxicity of MDR relevant drugs

We investigated the effects of tumor necrosis factor (TNF) , interferon (IFN) and interleukin-2 (IL-2) on themdr1 gene expression in four human colon carcinoma cell lines (LoVo, HT 115, SW 480, and LS 174T) at different times (8. 24, 48, and 72h). We found no significant changes inmdr1 expression after 8h and 24h of cytokine treatment in all four lines. After 48h and 72h, however, a marked reduction ofmdr1 expression in LoVo, HT 115, and SW 480 cells and an unaffected expression in LS 174T cells was observed. We examined whether the cytokine-mediated reduction ofmdr1 expression correlates to the multidrug resistance (MDR) phenotype. In those cell lines showing a decreasedmdr1 expression after a long-term cytokine pretreatment we found a dramatic enhancement of cytotoxicity of the MDR relevant drugs vincristine and doxorubicin, whereas LS 174T cells remained resistant. By contrast, the simultaneous application of cytokines and cytostatics caused no additive or synergistic effects. We conclude that in certain colon carcinoma cell lines a decreasedmdr1 expression caused by prolonged cytokine pretreatment correlates with an enhanced cytotoxicity of drugs susceptible to MDR as an MDR-overcoming effect.Abbreviations MDR multidrug resistance - TNF tumor necrosis factor - IFN interferon - IL-2 interleukin-2 - Vin vincristine - Dox doxorubicin - IC₅₀ inhibition concentration     

Reversal of multidrug resistance by novel cyclosporin A analogues and the cyclopeptolide SDZ 214-103 biosynthesized in vitro

It was shown that cyclopeptolide SDZ 214-103 (10 M) is more active in rhodamine-123 accumulation in actinomycin-d-resistant human lymphoma cells CCRF/ACTD400 than cyclosporin A (10 M), but equipotent in the doxorubicin-resistant Friend erythroleukemia cell line F4-6/ADR. In F4-6/ADR cells, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay showed comparable cytotoxic effects of doxorubicin at various concentrations in the presence of SDZ 214-103 and cyclosporin A. For the other novel cyclosporin A analogues minor multidrug-resistance-modulating potency was demonstrated. At equipotent modulating doses of verapamil (10 M) and cyclosporin A (10 M) in the MTT assay regarding doxorubicin cytotoxicity, cyclosporin A was efficient in the rhodamine-123-uptake assay while verapamil was not active when identical incubation times were used.Abbreviations MDR multidrug resistance - Pgp-170 P-glycoprotein with a molecular mass of 170 kDa - D-Hiv D-2-hydroxyisovaleric acid     

Activated cyclophosphamide: An enzyme-mechanism-based suicide inactivator of DNA polymerase/3′–5′ exonuclease

Summary DNA polymerase I fromE. coli can toxify activated cyclophosphamide (CP) by means of the 3–5 exonuclease activity associated with the enzyme. Acrolein and an alkylating moiety are released in the process. Preincubation of DNA polymerase I with activated CP for 15–60 min leads to an increasing inhibition of DNA polymerase activity, which can be prevented when preincubation of DNA polymerase I with activated CP is carried out in the presence of 5 AMP, a competitive inhibitor of the 3–5 exonuclease subsite of the enzyme. This demonstrates that toxification of activated CP by the 3–5 exonuclease subsite of DNA polymerase is a prerequisite for the inhibition of DNA polymerase activity. The kinetics and the degree of DNA polymerase inhibition suggest that the alkylating moiety rather than acrolein released from activated CP during toxification is responsible for the inhibition of DNA polymerase. DNA polymerase with associated 3–5 exonuclease activity has also been isolated from eukaryotic cells, and toxification of activated CP by such an enzyme (DNA polymerase from rabbit bone marrow) has been shown previously. Thus we suggest that toxification of activated CP by DNA polymerases/3-5 exonucleases present mainly in proliferating cells might lead to the specific alkylation of macromolecules involved in the cell proliferation process, such as the DNA polymerase subsite of these enzymes and probably also the DNA bound to the enzymes. The relatively high cancerotoxic selectivity and cytotoxic specificity of activated CP could be based on this specific enzyme-mediated alkylation.Supported by the Deutsche Forschungsgemeinschaft Bonn-Bad Godesberg     

Inhibition of protein kinase C in multidrug-resistant cells by modulators of multidrug resistance

We have evaluated the protein kinase C (PKC) activity in two series of cultured cell lines presenting the multidrug-resistance (MDR) phenotype and in the corresponding wild-type cells: the human KB 3.1, KB A1 and KB 8.5 cell lines, and the rat C6, C6 0.5 and C6 1V cell lines. We have observed an increase in PKC activity in the MDR cell lines of the KB cell lineage, proportional to their degree of resistance to doxorubicin. In contrast, the MDR cell lines of the C6 cell lineage presented no change (C6 0.5) or even decrease (C6 1V) in PKC activity; the basal level of PKC activity in C6 cells was, however, 50-fold higher than in KB 3.1 cells. We have tested, in these lines, the effect of four modulators of MDR: verapamil, cyclosporin A, quinine and S-9788, on doxorubicin acytotoxicity and on PKC activity. We observed that cyclosporin A and S-9788, which were the most active on MDR reversal, were able to inhibit PKC activity in the KB resistant lines as well as in all C6 lines, whereas verapamil and quinine had only marginal effects on PKC activity. The distribution of PKC isoenzymes was studied by Western blots. The PKC , and isoforms were increased in the KB resistant lines as compared to wild-type cells, which could account for the increase PKC activity we observed. In contrast, PKC and were decreased in C6 1V cells, as expected from the results obtained for total PKC activity, but we also noticed an important decrease in PKC in the C6 0.5 line. Our results suggest that an increase in PKC activity is not an absolute requirement for expression of MDR, provided that the basal level be high enough; and that some modulators may act on MDR, not only through direct P-glycoprotein interaction, but also through P-glycoprotein phosphorylation or expression. The distribution of PKC isoenzymes revealed that the modifications encountered between sensitive and resistant cells mainly concerned , and isoenzymes of PKC.Abbreviations PKC protein kinase C - MDR multidrug resistance     

GlutathioneS-transferase expression in malignant mesothelioma and non-neoplastic mesothelium: an immunohistochemical study

Expression of the glutathioneS-transferase (GST) subclasses , and was investigated immunohistochemically in 20 normal or hyperplastic mesothelium and in 57 malignant mesothelioma cases. These results were correlated with survival and also with P-170 glycoprotein expression. Nearly all the non-neoplastic mesothelium cases were positive for GST and . About half of the non-neoplastic cases were positive for . Twenty-nine (51%) malignant mesotheliomas were positive for at least one of the GST species; 21 (37%) showed immunoreactivity for , 18 (31.5%) for and 21 (37%) for . A total of 54 mesothelioma cases displayed immunoreactivity for the P-170 glycoprotein. For GST and GST, a statistical significance between expression and increased survival was found (respectivelyP=0.012 and 0.024) while for GST no significance was found. The results of this study demonstrate that expression of GST correlates positively with increased survival in malignant mesothelioma. It is also concluded that, in mesothelioma, GST and P-170 glycoprotein may contribute to the resistance to cytotoxic drugs frequently observed in these tumours. No correlation between GST and P-170 expression was demonstrated.Abbreviation GST glutathioneS-transferase     

“Atypical” multidrug resistance in human ovarian cancer cell line A2780 selected for resistance to doxorubicin (A2780 DX3)

Human ovarian cancer cells A2780, selected for resistance to doxorubicin (A2780-DX3), are crossresistant to various other topoisomerase-II-targeted drugs but not to vinblastine. The parental cell line was very sensitive to doxorubicin-, mitoxantrone- or etoposide (VP16)-induced DNA single-strand breaks, under deproteinizing conditions. In contrast, little or no DNA strand breakage was seen in resistant A2780-DX3 cells, even at very high concentrations, indicating a good correlation, with cytotoxicity. No significant alterations in cellular drug uptake were observed in DX3 cells. Further studies showed that the nuclei isolated from resistant cells were also resistant to mitoxantroneor VP16-induced single-strand breaks, indicating that nuclear modifications in resistant cells are responsible for this resistance. Catalytic activity in crude nuclear extracts from wild-type and DX3 cells was almost equal. However, an assay that specifically measures generation of 5-protein-linked breaks in³²P-labeled 3 DNA revealed that, DNA cleavage activity in nuclear extract from the DX3 cell line is profoundly resistant to a stimulation by VP16. These data indicate that stimulation of topoisomerase-II-mediated DNA cleavage is responsible for topoisomerase-II-targeted drugcytotoxicity rather than loss of normal topoisomerase catalytic function. These data support the hypothesis that A2780-DX3 cells display an atypical multidrug resistance.Abbreviations MDR multidrug resistance - SSB Single-strand break     

Production of interleukin-1β and tumour necrosis factor-α in patients with benign or malignant ovarian tumours

Summary To assess the role of interleukin-1 (IL-1) and tumour necrosis factor (TNF) in the physiological host defence mechanisms against malignancies, the production of these cytokines in sera, ascitic and cyst fluids and in the tumour tissues of patients with benign or malignant ovarian tumours was studied. IL-1 was found neither in the sera nor in the ascitic fluids of these patients. It was also virtually absent from the cyst fluid samples. However, a mean value of 790 pg IL-1/g tumour was found. Like IL-1, TNF was virtually absent in the serum samples. It was, however, detectable in the ascitic and cyst fluids and tumour tissues. The TNF concentrations were highest in the tumour tissues, with a mean level of 328 pg/g tumour. When comparing the level of IL-1 and TNF in patients with benign tumours to that seen in patients with malignant tumours, no differences in production were observed, regardless of the origin of the test samples. Our results indicate the production of IL-1 and TNF in patients with ovarian tumours. More importantly, the finding that the production of these cytokines in patients with benign tumours is similar to that in patients with malignant tumours supports the conclusion that the production of these cytokines is more a nonspecific indicator of an inflammatory process than a specific response to a malignant process.Abbreviations IL interleukin - TNF tumour necrosis factor     

Characterization of an established human,malignant, glioblastoma cell line (GBM) and its response to conventional drugs

A cell line, GBM, was established from a human malignant glioblastoma and was characterized with particular reference to its response to conventional drugs. The GBM cell line exhibited a 73±7 h doubling time in monolayer cultures. Experssion of glial fibrillary acidic and S-100 proteins was observed. Karyotype analysis of GBM cells at early passages revealed the presence of two near-triploid clones (A and B) with multiple chromosome rearrangements; a 100% frequency for clone B was observed in the established cell line. GBM cells had tumorigenic properties, since the s.c. injection of cultured cells into nude mice gave rise to slowly growing tumors. The morphology of GBM cells was retained during in vitro and in vivo passages, as judged by light microscopy. GBM cells were relatively resistant to most conventional drugs; among the tested drugs, only taxol exhibited a marked cytotoxic effect comparable to that found in cells of a different tumor type. GBM cells were found positive for the epidermal growth factor receptor, HER2-neu and P-glycoprotein by flow cytometry of cells labelled with monoclonal antibodies. In spite of the expression of relatively high -glutamyltransferase activity, the intracellular glutathione level was comparable to that of other chemosensitive tumor cells. This glioblastoma cell line is a suitable model for the identification and preclinical studies of new agents and provides an additional system to explore the molecular basis of the intrinsic drug resistance of glioblastoma.Abbreviations GFA protein glial fibrillary acidic protein - EDTA ethylenediaminetetraacetic acid - PBS phosphate-buffered saline - BSA bovine serum albumin - GSH glutathione - -GT -glutamyl transferase - WCP DNA probe whole-chromosome-painting DNA probe - SSC standard saline citrate - MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide - gp170 P glycoprotein - MDR multidrug resistance This work was supported in part by the Consiglio Nazionale delle Ricerche (Progetto Finalizzato Applicazioni cliniche della ricercy oncologica) and by the Associazione Italiana Ricerca sul Cancro     

Modulation of multidrug resistance by BIBW22BS in blasts of de novo or relapsed or persistent acute myeloid leukemia ex vivo

The phenylpteridine derivative BIBW22BS (BIBW22) is a potent modulator of multidrug resistance (MDR). We investigated BIBW22 in comparison to dexniguldipine and verapamil as modifier of MDR in blasts of de novo, relapsed or persistent acute myeloid leukemia (AML) in vitro. All patients with relapsed or persistent AML had been pretreated with idarubicin and cytosine arabinoside. The degree of MDR was determined by efflux kinetics of rhodamine 123 (R123), daunorubicin, and idarubicin measured by flow cytometry (FACS). A total of 51 patients with AML, 25 de novo and 26 relapsed or persistent, were investigated. While only 6 out of 25 de novo AML blast populations showed moderate efflux of R123 and daunorubicin, 17 out of 26 blast populations of relapsed or persistent AML had an efflux between 20% and 44% within 15 min ex vivo. This efflux could be significantly inhibited by 1 M BIBW22, 1 M dexniguldipine, or 10 M verapamil. For idarubicin we found an effusion of 40±9% within 15 min in all blast populations that could not be inhibited by the modulators. Clinically achievable drug concentrations causing only moderate side-effects are in the range of 0.5 M dexniguldipine and 3 M verapamil. Up to now, BIBW22 has not been investigated clinically. Thus, the potential toxicity of concentrations of 0.5–1 M BIBW22, sufficient for an optimal efflux inhibition ex vivo, is not known yet. We conclude from our ex vivo investigations in blast populations of de novo, relapsed or persistent AML that BIBW22 is a potent modulator of MDR.Abbreviations AML acute myeloid leukemia - MDR multidrug resistance     

Reversal of multidrug resistance by B859-35, a metabolite of B859-35, niguldipine,verapamil and nitrendipine

Summary It has been shown previously that verapamil and other calcium antagonists and calmodulin inhibitors can reverse multidrug resistance. We compared the potency of the dihydropyridine derivatives (4R)-3-[3-(4,4-diphenyl-l-piperadinyl)-propyl]-5-methyl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate-hydrochloride (B859-35), a metabolite of B859-35, niguldipine and (R)-nitrendipine to that of (RS)-verapamil in reversing multidrug resistance. The accumulation of the fluorescent dye rhodamine 123, which is transported by the P-glycoprotein, was determined by a flow cytometer. Multidrug-resistant human HeLa KB-8-5 and Walker rat carcinoma cells were incubated in the presence and in absence of the drugs indicated above. We found that 0.1 M B859-35 increases the accumulation of rhodamine 123 in multidrug-resistant KB-8-5 and Walker cells more effectively than 1 M (RS)-verapamil. In sensitive KB-3-1 cells addition of the drugs had no significant influence on the accumulation of rhodamine 123. In KB-8-5 cells, 10 nM Adriamycin caused a reduction of cell growth to 85% compared to untreated controls (=100%). If 1 M B859-35, B859-35 metabolite, niguldipine, verapamil or (R)-nitrendipine was added to 10 nM Adriamycin, growth reduction compared with untreated controls increased to 12%, 11%, 23%, 63%, and 82% respectively. The effect of 0.1 M B859-35 was a reduction in proliferation to 38%, that of 0.1 M verapamil to 72%. These data illustrate that B859-35, a compound with antitumor activity in several tumors, is at least ten times more potent than racemic verapamil in reversing multidrug resistance.Abbreviations B859-35 (4R)-3-[3-(4,4-diphenyl-1-piperidinyl)-propyl]-5-methyl-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylatehydrochloride - B859-35 metabolite, (4R)-3-[3,(4,4-diphenyl-1-piperidinyl)-propyl]-5-methyl-2,6-dimethyl-4-(3-nitrophenyl)-pyridime-3,5-dicarboxylatehydrochloride - MOPS 3-N-morpholinopropanesulfonic acid - MDRI human multidrugresistance gene - WR Walker cells resistant to alkylating agents and also multidrug-resistant     

Identification and characterization of novel mutations of the aspartoacylase gene in non-Jewish patients with Canavan disease

Canavan disease, an inherited leukodystrophy, is caused by mutationsin the aspartoacylase (ASPA) gene. It is most common among children of Ashkenazi Jewish descent but has been diagnosed in many diverse ethnic groups.Two mutations comprise the majority of mutant alleles in Jewish patients, while mutations in the ASPA gene among non-Jewish patients are different and more diverse. In the present study, the ASPA gene was analysed in 22 unrelated non-Jewish patients with Canavan disease, and 24 different mutations were found. Of these, 14 are novel, including five missense mutations (E24G, D68A, D249V, C152W, H244R), two nonsense mutations (Q184X, E214X), three deletions (923delT, 33del13, 244delA), one insertion mutation (698insC), two sequence variations in one allele ([10T>G; 11insG]), an elimination of the stop codon (941A>G, TAGTGG, X314W), and one splice acceptor site mutation (IVS1–2A>T). The E24G mutation resulted in substitution of an invariable amino acid residue (Glu) in the first esterase catalytic domain consensus sequence. The IVS1–2A>T mutation caused the retention of 40 nucleotides of intron 1 upstream of exon 2. The results of transient expression of the mutant ASPA cDNA containing these mutations in COS-7 cells and assays for ASPA activity of patient fibroblasts indicated that these mutations were responsible for the enzyme deficiency. In addition, patients with the novel D249V mutation manifested clinically at birth and died early. Also, patients with certain other novel mutations, including C152W, E214X, X314W, and frameshift mutations in both alleles, developed clinical manifestations at an earlier age than in classical Canavan disease.     

Effect of age on development of tumours in the intrasplenic ovarian grafts in ovariectomized rats

Summary Female rats at the age of 1, 3, and 14–16 months (young, adult and old groups respectively) were bilaterally ovariectomized and one of the removed ovaries was autoimplanted into the spleen. The total intrasplenic ovarian tumour incidence was equal in the rats subjected to the operation at 3 and 14–16 months (77.4% and 80.6% respectively) and tumours developed more frequently in rats exposed to surgery at the age of 1 month (94.5%),P<0.05. The incidence of Sertoli and Leydig cell tumours was increased and their latency was decreased in the old group in comparison to the adult one. In rats exposed to the operation at the age of 3 months, granulosotheca cell tumours developed more frequently than other tumour types, and in the young group thecomas were discovered more often than in both older groups. Dysgerminomas and luteomas were discovered only in intrasplenic grafts of rats of the young group. It is supposed that the differences in structure and proliferative activity of various ovarian tissues between young, adult and old rats at the moment of intrasplenic transplantation, as well as the differences in their response to gonadotropic stimulation play a significant role in the development of ovarian tumours of varied histogenesis in the intrasplenic ovarian grafts.     

Oligosaccharides accumulated in the bovineβ-mannosidosis kidney

Summary The phenotype of bovine-mannosidosis (-mannosidase deficiency), recently identified in Salers cattle, is similar to the caprine form of the disease (Abbittet al., 1991). This investigation was designed to characterize accumulated kidney oligosaccharides in bovine-mannosidosis. Oligosaccharides were extracted from the kidney of an affected Salers calf and purified by chromatographic techniques. The amount of accumulating oligosaccharides in 1 g of wet tissue was about 21µmol. Structures of derivatized oligosaccharides were characterized by high-performance liquid chromatography, mass spectrometry, methylation analysis and sequential exoglycosidase digestions. The major accumulating oligosaccharides were Man1-4GlcNAc and Man1-4GlcNAc1-4GlcNAc. Oligosaccharides accumulating in minor amounts were Man1-4GlcNAc1-4Man1-4GlcNAc, Man1-6Man1-4GlcNAc1-4GlcNAc and Man1-4GlcNAc1-4Man1-4GlcNAc1-4GlcNAc. As in caprine-mannosidosis, oligosaccharides with terminal-mannose residues and cleaved as well as uncleaved chitobiose linkages were identified in bovine-mannosidosis kidney. The accumulating oligosaccharides in tissue were thus identical in bovine and caprine-mannosidosis; however, the source of the novel oligosaccharides remains to be determined.     

Die β-Laktamase-Inhibitoren und ihre klinische Bedeutung

Zusammenfassung Zu den verschiedenen Möglichkeiten der Überwindung einer -Laktamase-bedingten Resistenz von Mikroorganismen gehört der Einsatz von Enzyminhibitoren, die selbst keine nennenswerte eigene antimikrobielle Aktivität aufweisen, jedoch in Kombination mit einem Breitspektrumantibiotikum vom -Laktamtyp synergistisch wirken. Auf diese Weise gelangen -Laktam-resistente Bakterien erneut in das Wirkungsspektrum von Substanzen wie Penicillin G oder Ampicillin, die aufgrund steigender Resistenzentwicklung in den letzten Jahren ihre therapeutische Effizienz zu verlieren drohen. 6--Bromopenicillansäure und die sogenannten Olivansäuren weisen eine bemerkenswerte Hemmpotenz gegenüber verschiedenen -Laktamasen auf. Die mikrobiologischen und bisher vorliegenden pharmakokinetischen Daten eines Penicillansäuresulfons, das ebenfalls signifikante Hemmeigenschaften verschiedener klinisch relevanter -Laktamasen besitzt, werden diskutiert. Von Clavulansäure, einem Stoffwechselprodukt vonStreptomyces clavuligerus mit -Laktamstruktur konnte ebenfalls gezeigt werden, daß es ein progressiver Hemmstoff der -Laktamasen vom Richmond-Typ II-V ist. Neben den bisher vorliegendenIn-vitro-Untersuchungen werden auch Ergebnisse klinischer Studien mit der Kombination Clavulansäure und Amoxicillin erwähnt.

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Inhibitors of -lactamases and their clinical significance

Summary One of the various possibilities of overcoming bacterial resistance due to -lactamase production is with enzyme inhibitors. These have no remarkable intrinsic antimicrobial activity, but act synergistically in combination with a broad spectrum antibiotic of the -lactam type. Thus -lactam resistant bacteria are once again within the antibacterial spectrum of substances like penicillin G or ampicillin, which have been in danger of losing their therapeutical effectiveness in recent years due to an ever increasing development of resistance. 6--bromopenicillanic acid and the so-called olivanic acids exhibit remarkable inhibitory properties against several -lactamases. Microbiological and pharmacokinetic data published recently on a penicillanic acid sulfone, which also shows significant inhibitory properties against several clinically relevant -lactamases, are discussed in this paper. Clavulanic acid, a recently discovered product ofStreptomyces clavuligerus with a -lactam structure, acts as a progressive inhibitor of Richmond type II-V -lactamases. In addition to microbiological and enzyme-kineticin vitro data, results of clinical studies with the combination clavulanic acid and amoxicillin are summarized.

     

Early prognosis after thrombolysis: value of exercise radionuclide ventriculography performed on anti-ischaemic medication

We examined the prognostic value of exercise radionuclide ventriculography (RNV) performed on anti-ischaemic medication following thrombolysis. Within 3 months of thrombolysis for first myocardial infarction, 31 medically treated patients with revascularisable but non-critical and minimally symptomatic coronary disease had left ventricular ejection fraction (LVEF) measured by first-pass exercise RNV. This was first performed off treatment and then repeated within 4 weeks on patients' regular medication. Follow-up at 12 months post-thrombolysis showed that 5 patients (Group I) had suffered significant recurrent symptoms (worsening angina requiring revascularisation in 3, unstable angina in 1, reinfarction in 1), but 26 remained well (Group II). Both groups were similar in age, post-thrombolytic severity of coronary disease, exercise LVEF whether off (39%vs 43%) or on medication (43%vs 44%), and change in LVEF with exercise (LVEF) off medication (–11%vs –3%). However, on medication, there was a significant difference in mean LVEF between Groups I and II (–11%vs+5%,P=0.0008, 99% confidence interval=4 to 26%).Thus, following thrombolysis, an abnormal LVEF despite anti-ischaemic medication may identify patients at risk of significant early recurrent ischaemia. Post-thrombolysis prognostic testing by exercise RNV may therefore be of greater value when performed on rather than off medication.     

Treatment of clinical stage I testicular cancer and a possible role for new biological prognostic parameters

Three different treatment strategies for patients with stage I non-seminomatous testicular cancer are available that will all result in long-term survival in more than 98% of the patients: a wait and see strategy with follow-up and chemotherapy in cases of tumour progression, retroperitoneal lymphadenectomy, with or without application of systemic chemotherapy, in cases of retroperitoneal metastases (pathological stage II disease) or primary adjuvant chemotherapy following inguinal orchiectomy. Each treatment strategy is associated with specific side-effects. In several studies histological characteristics of the primary tumour, particularly the presence of vascular invasion and of embryonal carcinoma cells, have been demonstrated to be significant prognostic factors for the risk of occult retroperitoneal metastases in patients with stage I disease. In addition, new biological prognostic factors determined by flow cytometry, cytogenetic analysis or molecular-biological DNA or RNA analysis have been investigated, among which alterations of thep53 tumour-suppressor gene may represent a promising new prognostic factor. Although alterations ofp53 gene expression seem to be associated with advanced tumour stage and may predict retroperitoneal metastatic disease, the independent role of these molecular genetic alterations needs to be prospectively studied. Currently a risk-adapted treatment strategy based on the histological criteria of vascular invasion and the presence of embryonal carcinoma can be used to stratify patients into a high- and low-risk group with respect to tumour progression. While primary-nervesparing retroperitoneal lymphadenectomy or adjuvant chemotherapy with two cycles of platinum, etoposide and bleomycin may be appropriate for patients with a high risk (above 40%) for tumour progression, a wait-and-see strategy can be used for low-risk (less than 15% risk of progression) patients. Molecular investigations of prognostic factors may be able to improve further the stratification of patients into these different risk categories.Abbreviations RLA retroperitoneal lymphadenectomy - SCF stem-cell factor     

Inhibition ofO 6-alkylguanine-DNA alkyltransferase and DNase I activities in vitro by some alkylating substances and antineoplastic agents

Summary The specificities of the DNA repair enzymeO ⁶-alkylguanine-DNA alkyltransferase from brain and liver cells of the chick embryo and of DNase I were demonstrated in vitro by their response to substrate DNA pretreated with monofunctional alkylating agents of differentO ⁶-guanine alkylating ability and some antineoplastic agents. Treatment of DNA with ethidium bromide, Hoechst 33258, doxorubicin, Fe²⁺/bleomycin, and suramin resulted in a dose-dependent diminution of alkyltransferase activity (DE₅₀ 5 g/ml, 15 g/ml, 5 g/ml, 5 g/ml, 100 g/ml, respectively). Apart from bleomycin, comparable results were obtained with DNase I. Thermal denaturation of the substrate DNA reduced both alkyltransferase and DNase I activity. No effect was seen with X-irradiation. Cisplatin decreased only DNase I activity. Some topoisomerase II and/or gyrase inhibitors remained without significant effects on the alkyltransferase reaction whereas DNA catabolism by DNase I was diminished in a dose-dependent manner (DE₅₀ between 6.5 and 19 g/ml).Abbreviations AT alkyltransferase - BB bisbenzimide - EB ethidium bromide - DOX doxorubicin - CDDP cis-diamminedichloroplatinum (II) - MMS methylmethanesulphonate - EMS ethylmethanesulphonate - MNU methylnitrosourea - ENU ethylnitrosourea     

Changes in expression levels of genes involved in fatty acid metabolism: upregulation of all three members of the PPAR family (α, γ, δ) and the newly described adiponectin receptor 2, but not adiponectin receptor 1 during neonatal cardiac development of the rat

During neonatal cardiac development, the heart changes its substrate preference from glucose to fatty acids. The aim of this study was to investigate the changes in mRNA expression levels of genes involved in the control of cardiac fatty acid metabolism in the transition from neonatal to adult life. Methods mRNA expression levels for peroxisome proliferator activated receptor (PPAR) , and , PPAR co–factor 1 and (PGC–1 and ), 9–cis retinoc–acid–activated receptor , and (RXR , , ), 5–AMP activated protein kinase (AMPK) 1 and 2, adiponectin receptor 1 and 2 (AR 1 and AR 2) were measured in heart tissue of neonatal 0–day, 7–day and 21– day old rats. Results mRNA expression of all three members of the PPAR family were upregulated significantly from day 0 to day 21 ( +117%, +133%, +203%). In addition, m–RNA expression of all RXR isoforms increased from day 0 to day 7 ( +125%, +69%; +41%). AR 2 exhibited a small but significant increase in mRNA expression (+ 46%). Conclusions We were able to demonstrate for the first time that in addition to PPAR, also PPAR and , as well as all RXR isoforms and AR 2 are upregulated in the heart during neonatal development.Drs. Steinmetz and Quentin contributed equally to this publication     

γ-Glutamyl transpeptidase-cellular expression in populations of normal human mononuclear cells and patients suffering from leukemias

The expression of the ectoenzyme-glutamyl transpeptidase (EC2.3.2.2.,GT) was investigated by flow cytometry on populations of peripheral blood mononuclear cells (PBMC) from healthy subjects and patients suffering from several types of leukemia before and under chemotherapy. In unstimulated PBMC, 28% of these cells were found to beGT positive. The highest expression was measured on monocytes (CD14/GT⁺ cells: 60%). Within the subsets of T lymphocytes (CD3/GT⁺ cells: 18%) we saw no clear differences between CD4⁺ and CD8⁺ cells. B lymphocytes, NK cells, and activated cells showed low expressions (up to 10%). Treatment of PBMC with mitogens, -IFN, IL-2, and GM-CSF did not affect the enzyme expression on normal mononuclear cells (MNC). However, a rapid increase of yGT⁺ cells was found in the presence of glutathione (GSH) and n-acetyl cysteine (nAC), particularly on monocytes, B cells, and NK cells. Comparing 40 healthy subjects and untreated patients suffering from leukemias, a significantly higher expression ofGT⁺ cells in the total MNC populations (B-CLL: 57%, CML: 62%GT⁺ cells) was observed in B-chronic lymphocytic leukemia (B-CLL) and chronic myelogenous leukemia (CML), whereas other leukemias did not show clear differences. Most interestingly, theGT expression was diminished in all populations of CML cells after 5 h of incubation in the presence of 10 units/ml IFN-. These data suggest a possible protective role ofGT in MNC and a regulatory function of this enzyme in the development of CML.     

Aspartic acid at position 57 of DQβ chain does not protect against Type 1 (insulin-dependent) diabetes mellitus in Japanese subjects

Summary HLA DQ chain, in particular amino acid at position 57, has been reported to contribute to susceptibility and resistance to Type 1 (insulin-dependent) diabetes mellitus in Caucasians. Resistance has been proposed to be conferred by aspartic acid at this position. To ascertain the association of HLA DQ and DR genes with Type 1 diabetes in Japanese subjects, ten Japanese Type 1 diabetic patients were investigated at DNA level. Genomic DNA was amplified by polymerase chain reaction, and dot blot analysis was carried out using the amplified DNA with allele specific oligonucleotide probes. All patients had aspartic acid at position 57 of at least one of their two DQ chains, and there was no significant difference of amino acids at the same position of DR chain in patients compared to control subjects. These data indicate that the protective role of aspartic acid at position 57 of DQ chain is less significant in Japanese compared with Caucasian subjects.