Abstracts

of

Oral Presentations

P U L M O N A R Y D I S T R I B U T I O N O F T H I O P U R I N E N U C L E O T I D E S IN L U N G - T R A N S P L A N T P A T I E N T S

R.Bou l i eu . A .Leno ir . J . F . M o r n e x

Azathioprine, a thiopurine compound that exhibits immunosuppressive activity is current ly used with cyclosporine and cort icosteroid in combined immunosuppmssive protocols in organ transplantation (1). Azathioprine undergoes a complex metabolic pathway and its pharmacodynamic activity is considered to depend on the formation of intracellutar thiopurine nucleotides (2). Despite the extensive use of azathioprine in lung transplantation, data on diffusion of thiopurine nucleotides into the lung are not available. Therefore, we investigated lung distribution of thiopurine nucleotides of 6-thioguanine (6TGN), 6-mercaptopudne (6MPN) and 6-thioxanthine (6TXN) in eigth lung-transplant patients under azathioprine therapy.

Thiopurine metabolites were determined in cellular fraction of bronchoalveolar fluid (BALl =) and in red blood cells from patients receiving 2.5 mg/kg daily of oral azathioprine. Nucleotides of 6TG, 6MP and 6TX were analysed by high performance liquid chromatography. Sample treatment consisted of PCA deproteinisation of cell suspension and hydrolysis of nucleotides to their free bases by heating of the PCA extract (3). In cellular fraction of BALF, 6TGN were recovered in three patients at concentrations ranging from 827 to 1667 pmol/108 cells. In three other patients, the thiopurine nucleotide found was 6MPN at concentrations ranging from 240 to 330 pmol/108 cells and in two patients, no thiopurine nucleotides were detectable. In red blood cells, 6TGN was recovered in all patients in the concentration range of 9 to 121 pmol/lO 8 cells. In five patients, 6MPN was present at concentrations from 2 to 138 pmol/I08 cells.

These preliminary results show that thiopurine nucleotides of 6TG and 6MP that are considered to be the active metabolites can diffuse into the lung at significant levels. Further investigations are needed to define the relationship between thiopurine nucleotide levels recovered in lung and the occurence of pulmonary rejection.

I R.L. Simmonds el al,Transplant. Proc.,18 (1986) 76. 2 G.B. Elion, G.H .Hitchings, In O. Eichler et al (eds), Handbook of

Experimental Pharmacology, NY, t975, 404. 3 R. Bodlieu et al, J. Chromatogr., in press.

Laboratoire de Pharmacie Clinique, Institat des Sciences Pharmaceutiques el Biologlques, Lyon, France - Service Pharmaceutique el Service de Bronchopneumologie, H6pital Cardiologique, Lyon, France.

MECHANISTIC STUDY ON THE ANTIVIRAL AND CYTOSTATIC EFFECTS OF 5-THIEN-2-YI~ AND 5-FURAN-2-YL-SUBSTITLYFED PYRIMIDINE NUCLEO- SIDE ANALOGUES.

C. Bohman ~', J. Balzarini'}" P .Wigerinck �89 A. Van Aerschot #, P. Herdewiin +, ~rkk~req- F

A novel group of pyrimidine nucleoside analogues, containing a furan-2-yl- or a thien-2-. yl- moiety at C-5 of the uracil or cytosine ring have been synthesized (1). The compounds markedly inhibit herpes simplex virus type 1 (HSV-1) repllcatinn in cell culture. The halogenated 5-thian-2-yl-substituted 2'-deoxyuridine (dUrd) and I-g-D- ribofuranosyluracil (araU) derivatives show a 50%-effective concentration (EC50) ranging between 0.10 and 0.19 I~M. These values are only 1.5- to 3-fdld higher than recorded fur (E)-5-(2-bromovinyl)-dUrd (BVDU), an antiherpetic compound that has proved efficacious in the treatment of HSV-1 infections in humans. The affinity of the test compounds for HSV-1 thymidine (dThd) kinase (TK) was evaluated. HSV-1 TK was purified approximately 50,000-fold from marine mammary carcinoma (FM3A) ceils deficient in cytosol thymidine kinase and transfected with the HSV-I TK gene (FM3A TK'/HSV-1 TK+). 5-Furan-2-yl-dUrd, 5-thien-2-yl-dUrd, and 5-(5-bromothlen-2-yl)- dUrd emerged as the most potent inhibitors of thymidine phosphorylation by HSV-1 TK. Their 50%-inhibltory concentrations (IC50) ranged from 2.4 to 3.5 p.M. The compound concentrations required to inhibit thymidine phosphorylatinn by cytosot tbymidine kinase were at least 55- to more than 200-fold higher. However, we found no correlation between the inhibitory effect of the test compounds on HSV-1 replication and their inhibitory effect on HSV-1 TK catalyzed phosphorylation of dThd. These data suggest that the presumed target for the antiviral action of the compounds, that is HSV- 1 DNA polymerase, may have a different structure-activity relationship (SAR) for these compounds than HSV-I TK. The effect of the compounds on cell proliferation, incorporation of DNA_ precursors into trichloroacetic acid-insdlublc material, and tritium release from [5-3H]dCyd in wad-type FM3A and FM3A TK'/HSV-1 TK § ceils was also investigated. FM3A TK'/HSV-1 TK + ceil growth was strongly inhibited in the presence of either 5-furan-2-yt-dUrd, 5-thien-2-yl-dUrd or 5-thien-2-yl-dCyd (the IC50 concentrations varying from 1.4 to 4.6 I~M), whereas growth of the corresponding wild- type FM3A cells was not inhibited at concentrations up+ to 500 ~M. The cytostatic action of these compounds for FM3A TK-/HSV-1 TK cells could be ascribed to preferential phosphorylation by the virus-encoded TK to their monophosphate form, followed by inhibition of cellular thymidylate symhase. In this respect, the test compounds behaved similarly to BVDU (2,3). However, in contrast with BVDU that had previously been shown to be an effective substrate for dThd pbosphorylase (4), we found that 5-furan-2-yl-, 5-thien-2-yl-dUrd and 5-thien-2-yl-dCyd showed poor, if any hydrolysis by dThd phosphorytase. 1. P .Wigerinck et al. J. Med. Chem. 34 (199t) 2383 2. J. Balzarini et al. Mol. Pharmacol. 32 (1987) 410 3. J. Balzarini and E. De Clercq. Methods Find. Exp. CAin. Pharmacol. 11 (1989) 379 4. C. Desgranges etal . Biochem. Pharmacol. 32 (1983) 3583

+Laboratory of Experimental Chemotherapy, and %Laboratory of Pharmaceutical Chemistry, Rega Institute for Medical Research, Katholieke Universiteit Leuven, B- 3000 Leuven, Belgium

ADENINE NUCLEOTIDE ~ETABOLISM IN PRIMARY RAT NEURONAL CULTURES

5. Brosh, E. Zoref-Shani, O- Sperlin~, E- Danzi~er, Y. Sidi,

The metabolic fate of prelabeled adenine nucleotides (AdRN) was studied in immature (~ d old) and mature (8-14 d old) primary rat neuronal cultures. The label in AdRN decreased with time, appearing in acid insoluble derivatives and in hypoxan%hine. The combined addition, for 4 h, of 2'-deoxycoformycin and of S'-amino-S'-deoxyadenosine resulted in both cultures in a decrease of about 6M in the label in AdRN, associated with a similar increase in the label in adenine and adenosine. Addition of alanosine resulted in both cultures in a 2-3 fold increase in the label in hypoxanthine and inosine, associated with a significant decrease of the labellng in AdRN. Enhanced degradation of AdRN was accomplished by incubation for 2 h with iodoacetate and antimycin. Under such conditions, the immature cells lost 58 ~ and the mature cells 74 ~ of the label from AdRN, most Of it to bypoxanthine and inosine, but also to adenosine. Coaddition of 2'-deoxycoformycin, decreased markedly the label in bypoxanthine and inosine, and increased the labeling of adenosine and adenine. In both cultures, the enhanced degradation was associated with a complete halt of transfer of label from AdRN to the acid insoluble fraction. The results demonstrate that in the cultured neurons, AdRN are metabolised to acid insoluble derivatives, or degraded to hypoxanthine. Under physiological conditions AdRN are degraded through both dephosphorylation and deamination of AMP. Enhanced degradation of AMP proceeded mainly through dephosphorylation in immature cultures, and equelly via both pathways in the mature cells. Under physiological conditions, the flux of label from AdRN to the acid insoluble derivatives is more intense in the immature neurons. The adenosine-AM~ cycle operates in the cultured neurons. Both arms of the adenine nucleo%ide cycle (IMP to AMP and AMP to IMP) are operating in the cultured neurons, but, the intensity of this cycle has not yet been clarified and it is not known yet if this cycle has any slgnificant role in this tissue, other than synthesis and degradation of AMP.

Dep%s. of Clin. Bfochemistry and of Medicine D, Beilinson Hed. Center, Petah Tikva and Dept. Of Chemical Pathology, Sackler School of Medicine, Tel Aviv University, Israel.

l ' ha rma~/ I I b d d L ~ Sc wm r

Vo,o. .~ ,s N . . ,~9~ F 7

DETECTION OF POINT MUTATIONS AT THE HUMAN HPRT-LOCUS USING THE RAPID SINGLE STRAND CONFORMATION POLYMORPHISM (S$CP) ANALYSLS

Renate Buroemeister. Elke Rotzer, Wolf Gutensohn

Comp le te de f i c iency of HPRT causes the Lesch-Nyhan syndrome (LNS) which is character ized by hyperur icemia, menta l retardat ion, choreoathetosis, and compuls ive self4"nutilation. Partial de f i c iency of HPRT leads to a severe form of gout and nephtolithiosis.

Here w e descr ibe three mutat ions in three nonrelated families previously studied by direct sequenc ing of PCR products of the human HPRT gene.

* Pat ient IJ shows an insertion of one G in exon 3 of the HPRT gene. He represents a case of the classical Lesch-Nyhon syndrome. There is no residual act ivi ty of HPRT in his erythrooyte lysate. His mother is heterozygous for the disease whilst his sister shows no mutations, * Pat ient GS has a muta t ion in exon 7 (CGA - > TGA, Are --> Stop). He shows a partial HPRT-deficlency, a residual act iv i ty o f 1,8% of normal is found in his erythrooyte Iysate. His mo the r shows no rt,,utation, so it is assumed that this is a case of a new mutat ion. * Pat ient KM has a point muta t ion in exon 2 (ATr --:, ITT, lle --> Phe) and he shows a partial HPRT-deticiency. His residual act iv i ty of HPRT is 2.7% of normal, His mother is heterozygous. Patients GS and KM represent cl inical borderl ine cases, including not only the symptoms of gout, but also some of the neurological symptoms of LNS lacking the the typical autoaggress ive behavior .

For the rapid ar id simple de tec t ion of point mutat ions we used the single strand con fo rmat ion polyrnorphlsm (SSCP) analysis. Single s t randed DNA molecules ore c a p a b l e of forming unique secondary structures whose nature is dependen t upon their base sequences, The type and deg ree of secondary structure format ion in turn determines e lec t rophoret ic mobil i ty under nondenatur ing conditions. Mutat ions within a g iven DNA segment alter this mobil i ty and the "mobil ity shift" may be de tec ted on high resolution nat ive PolyacryJamid gels.

We demons t ra te that SSCP analyf~s c a n reliably and reproducibly identit iy the mutat ions we had found previously in three exons of the human HPRT gene. All mutat ions d e t e c t e d by d i rect DNA sequencing were also de tec l ed by SSCP in all p robands tested. Individuals homozygous and heterozygous for the mutat ions in this X ch romosoma l disease are olearly distinguishable. The simpl~i ty of this techn ique together with its speed and reliability c o m m e n d it for rout ine use in molecu lar diagnost ic medic ine.

Institut fGr Anthropolog ie und Humongenet ik, Goethestr. 31, W-8000 MOnchen 2, FRG

THE iSOLATED RAT SUPERIOR CERVICAL GANGLION (SCG) AS A MODEL TISSUE FOR THE STUDY OF NEURONAL PYRIMIDINE RECEPTORS.

G.P.Connollv & P.J. Harrison.

In the cardiovascular system there is some evidence in favour of the presence of extracellular receptors that are activated specifically by uridine 5'-triphosphate (UTP) rather than adenosine 5'-triphosphate (ATP) i.e., pyrimidinoceptors, however it is unknown if these receptors also exist within the nervous system. We bare investigated this possibility using methods described before 1.

SCG were placed in a chamber and perfused with physiological salt solution+(PSS) at 25+ I ~ 7.4 equilibrated with 5% CO,V95%O 2 containing 2raM K and 0. I mM Ca 2 4 . The d.c. potential between the ganglion and the postganghomc nerve was recorded across a greased barrier, Adenosine and pyrimidines were added to the perfu~ate for 2 rain and other drugs for 1 rain. Responses in the presence of atropine (2aM) or suramin (300,aM) were determined after I5 or 30 rain preincubation.

On the same ganglia UTP was more potent thatl a/bMeATP in producing concentration -dependent depolarisatioos. Depolarisations caused by aBMeATP were abolished by suramin, whereas depolarisation caused by UTP were potentiated, and hyperpolarisations to adenosine, depolarisatioas to muscarine, DMPP or K + remained unaltered (Table 11. In the presence of suramirr, atropine abolished depolarisations caused by muscarine (100nM, 276 _+ 33~V cf 6 _ 7~tV, n=4, P<0.01) but not those caused by 100~M UTP (263 -+ 67,aV cf200 4- 41/xV, n=4).

Effect of suramin on thexesoonse (uV, me,.a~t +/- s.e.mean (4-9 SCGI. .= P < 0_01.*~* P < 0.01. Paired t-test/to denoladsing and h yoemolarisin~_aeooists.

A~anist ~/bMeATP UTP Adenosine Muscarine DMPP K + Tceatment 100ttM 100~M I00#M 100nM 10uM 3mM CONTROL 76+I1 216_+38 -1405:35 231_+32 1214-27 136+_33

SURAMIN -5_10"** 2834-44"*-161• 268+29 139_+35 1144-28

The inability of atropine to alter depoiarisation to UTP indicates that UTP did not depolarise ganglia by releasing acetylcholine to activate muscarinic receptors or activate muscarinic receptors directly. The selective antagonism of the response to uBMeATP and not UTP by suramin is consistent with the presence of both P2- purinoceptors and receptors for UTP. Our results provide evidence for the presence of pyrimidinoceptors in the nervous system and suggest that the rat SCG may be a useful model system for studying these receptors.

1. G.P. Connolly & T.W. Stone. J. Auton. Pharmacol., 13 (1993) 213-224.

Department of Physiology, University College London, Gower Street, London. U.K.

DIHYDROPYRIMIDINE D E H Y D R O G E N A S E ACTIVITY IN H U M A N PERIPHERAL BLOOD M O N O N U C L E A R CELLS AND LIVER:

POPULATION CHARACTERISTICS, NEWLY IDENTIFIED DEFICIENT PATIENTS, AND CLINICAL IMPLICATION IN 5-FLUOROURACTL

C H E M O T H E R A P Y

R.B. Diasio, Z. Lu,. and R. Zhan~

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in catabolism of 5-fluorouracil (FUra), one of the most widely used anticancer drugs. Previous studies from our laboratory 1,2 demonstrated the clinical importance of DPD in cancer patients particularly in those with DPD deficiency who experience severe FUra toxicity (including death) following FUra treatment 3,4. We now suggest that measurement of DPD activity may be useful in routine screening of cancer patients prior to FUra treatment and describe the following serial studies. First, development of a sensitive, accurate, and precise DPD assay and a storage method to stabilize DPD activity, permitting large scale DPD screening in cancer patients. Second, demonstration of a normal distribution (Gaussian distribution) of human DPD activity from peripheral blood mononuclear cel!s (PBM-DPD) in a population study, l~aselines (mean -.+ SD) for PBM-DPD with fresh and frozen samples were 0.425 +- 0.124 and 0.189 *-. 0.064 nmol/min/mg protein, respectively. The 95% and 99% distribution ranges for both fresh and frozen samples were also determined, providing criteria for detection of DPD deficient patients. Third, identification of 9 new patients with profound or partial DPD deficiency. Fourth, determination of a baseline for human liver DPD activity, which was shown to be 0.360 -,-0.182 nmol/min/mg protein (frozen samples). Fifth, preliminary evaluation of liver DPD from deficient patients. Low liver DPD activity in 2 deficient patients correlated with low PBM-DPD activity. Using a polyclonal antibody demonstrated decreased DPD protein in the liver cytosol from DPD deficient patients compared to normal subjects. These results may be useful in improving the effectiveness and/or lessening the toxicity of FUra chemotherapy. (Grant NCI CA 40530).

1 G.D. Heggie, et al. Cancer ges . 47 (I9871 2203. 2 B.E. Harris, et al. Cancer Res. 50 (19901 197. 3 R.B. Diasio, et al. J. Clin. Invest. 81 (19881 47. 4 B.E. Harris, et al. Cancer 68 (19911 409. s Z. Lu, et al. J. Biol. Chem. 267 (1992) 17102.

Departments of Pharmacology and Medicine, Division of Clinical Pharmacology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A.

FENOFIBRATE: A LIPID LOWERING DRUG WITH URICOSURIC PROPERTIES

U. Gresscr,

Fenofibrate, a fibric acid derivate, is used for the treatment of hyperlipoproteinemias. In addition to its lipid lowering properties feaofibratr shows a uricosoric effect. In healthy volunteers feoofibrate lead to a 2-fold, benzbromarone t9 a 4-fold increase of uric acid clearance I. In hyperuricemic outpatients the decrease in plasma uric acid over a period of one year was 30 % of the initial value 2. Other authors found a de- crease of plasma uric acid of 20 %3, 4 In comparison hczafibrate and placebo had no uricosuric effect 3. Most of these studies were performed to measure the lipid lowering effect of fenofibrate and therefore give no detailled informations on extent and course of time of the uricosuric effect of fenofibrate. Aim of our study was to get detailled informations on these points in comparison to a well-known uricosaric drug. We compared the uricosurie and lipid lowering effects of a single daily dose of feno- fibratc (200 mg, micronized, Knoll AG) with benzbromaxone (100 rag, ratiopharm) in a cross-over study with 7 healthy volunteers on a low purine dict. Our results confirm the observations of a distinct uricosoric effect of fenofibrate. In 7 healthy volunteers a single dose of 200 mg fenofibrate lead to an average additional renal uric acid excre- tion of 204 mg Coenzbromarone: 376 rag) within 14 hours after drug ingestion. So fenofibrate lead to 54 % of the additional renal uric acid excretion in comparison to benzbromarone in the same volunteers. If we include that the normal dally dose of beazbromarone would be 80 rag, the relative uricosurie effect of feoofihrate is more impressive. Between hyperurieemia and hypertriglyceridemia there is a significant correlation (p<0,001) 5. Therefore many patients need a uric acid lowering and a lipid lowering treatment. Fenofibrat preferentially is a lipid lowering drug, but with its clear uricos- uric properties it could be a good possibility in many patients with both metabolic dis- eases. The uricosaric effect of fenofibrate is large enough to normalize uric acid in most patients with hyperlipidemia and hyperuricemia. With its combined lipid lowering and uricosuric properties fenofibrate will help to shed light on the pathophysiology of the correlation between hyperlipidemias and hy- peruricemia. We give detailled informations on the influence of fenofibrate and benzbromarone on plasma uric acid, renal uric acid excretion, triglycerides, HDL- and LDL-cholesterol.

1. J.P. Desager et at., J Clin Pharmacol 10: 560-564, 1980 2. C. Harvengt et at., Artery 7 (I): 73-82, 1980 3. M.D. Bestow et at., Metabolism 37: 217-220, 1988 4. J. SteinmetZ et at., Clinica Chimica Acta 112: 43-53. 1981 5. B.S. Gathof et at., Adv Exp MeAl Bioi 309A: 231-234, 1991

Medizin. Poliklinik, Universit~t Miinchen, Pettenkoferstr. 8a, 80336 Mfinchen, FRG

Vo~um~ 15 Nr. a I~93

THE ACTIVE SITE OF HUMAN ECTO-5'-NUCLEOTIDASE. DELINEATION OF ESSENTIAL AMINOACID RESIDUES BY SITE

DIRECTED MUTAGENESIS AND AN ATTEMPT TO DISSECT CATALYSIS FROM OTHER FUNCTIONS OF THE MOLECULE.

W. Gutensohn, R.Resta, L.F.Thompson

Recent research has yielded a somewhat clearer picture of the biological function(s) of ecto-5'-nucleotidase (5'-NT). As an enzyme it may contribute to the removal of potenti- ally toxlc purine metabolites in the neighborhood of dying cells or regulate critical ligand concentrations for puri- nerglc receptors. In addition 5'-NT was shown to act as an accessory molecule in T-lymphocyte activation and here it is less clear, whether the catalytic property of the mole- cule is required or rather its particular type of membrane anchorage via glycan-phosphatidyl-inositol. We attempted to dissect such possible functions. Starting from 5'-NT cDNA and guided by indirect evidence from chemical modification and sequence comparisons the codons for three conserved hl- stidlne residues were changed to alanine codons. Wild type cDNA and mutant constructs were expressed in the 5'-NT-ne- satire T-lymphoblastoid cell line Jurkat. Surface expres- sion of 5'-NT in the transfectants was monitored by immunofluorescence using monoclonal antibodies (mAbs) and was seen in the wild type as well as the mutants. Enzyme activity on the other hand was high in the wild type trans- fectants, but was completely abolished in the 3 different His to Ala mutants. Using the same mAbs it could be clearly shown that mutant 5'-NT devoid of enzymatic activity could still function in signal transduction during stimulation of the Jurkat transfectants and thus this property could be separated from catalysis. The roles of the 3 different His residues in 5'-NT enzymatic activity-have still to be defi- ned more precisely. They might be involved: i) in the cata- lytic reaction proper as acceptor of phosphate in a short- lived intermediate ii) as an essential constituent of a nucleotide binding site and iii) in zinc coordlnation.

Illstitute of Anthropology and Human Genetics, University of Munich, Goethestr. 31, D 8000 Munich 2, Fed. ReD.Germany. Oklahoma Medical Research Foundation, 825 N.E. 13th Street, Oklahoma City, OK 73104, USA.

MAGNETIC RESONANCE IMAGING ENLARGES THE DIAGNOSTIC POSSIBILITIES IN HYPERURICEMIA AND GOUT

I. Kamilli I , U. Gresser 1, R. Rauch 1 , D. Hahn 2

In patients with chronic gout uric acid deposition appears in bones near to joints, in the synovial membrane, in bursae, tendon sheats and other bradytrophical tissues. Whereas bone tophi and superficial tophi are well accessible, tophi in non super- ficial tissues, e.g. heart valves, eyes, spine and peripheral nerves, can not be iden- tified and we find them only surgically or by autopsy. The purpose of this study was to investigate the valuation of scintigraphy and magnetic resonance imaging (MR/) in comparison to clinics and conventional radiological procedures for de- monstration of uric acid deposition.

Twentyfive patients with chronic gout followed in the Medizinische Poliklinik par- ticipated in this study. In every patient (n=25) radiographs of the feet, big toe, ankle joints, knees, hands, wrists and elbows (joint region=350) and an early (5 min) and late (2 hours) static scintigraphy (99mTcPP) were obtained. In 9 patients, 7 feet, 6 hands and one elbow a MRI was done. Coronal, axial and sagittal T1 and T2 weighted MR images with and without Gd-DTPA were obtained. We compared the findings in MRI, clinical evaluation, x-ray and scintigraphy in every joint region. In MRI in 14 (100%) joint regions soft-tissue tophi were found, whereas in clinical evaluation and x-ray only in 8 joint regions tophi were registra- ted and in scintigraphy only in 11 joint regions higher concentrations of activity were found. In MRI tophi appear in T I weighted images as hypotense inhomoge- nous structures and in T2 weighted images as hyperintense structures. With Gd- DTPA tophi appear in T1 weighted images as structures enhancing the contrast medium; in larger tophi there is only a peripheral enhancement and a central zone with a tow signal intensity. In contrast to all the other diagnostic possibilities only in M R/bone marrow edema are obtained as a sign of inflammation.

MRI is the only method to mark off the complete extension of tophi. Therefore MR/en la rges the diagnostic possibilities in hyperuricemia and gout, especially the diagnosis of the extend of tophi in non superficial tissues, e.g. eyes, spine, skull- base and perineural tissues such as the carpal tunnel.

I Medizinische Poliklinik der LMU-Mftnchen, Pettenkoferstr. $a, 80366 Mfinchen, Germany 2Radiologische Polikliaik der LMU-Mfinchen, Pettenkoferstr. 8a, 80366 Mtnchem Germany

AICARIBOSIDE INHIBITS GLYCOLYSIS IN HEPATOMA CELLS

F. Javaux. MF. Vincent. G. Van den Bemho

AICAriboside, the nucleoside corresponding to AICAR (ZMP), an intermediate of de nove purine synthesis, inhibits glycolysis in isolated rat hepatocytes (t). Since many tumour cells depend on a high glycotytic rate (2) to provide the energy required for proliferation, the effect of AICAriboside was investigated in the rat hepatoma cell line FTO-2B.

In intact FTO-2B. cells, addition of 250 l.tM AICAriboside resulted in the accumulation of 5 mM ZMP. Production of lactate with 10 mM glucose was inhibited by 65% within 2 h. Half-maximal inhibition was reached with 50-100 p.M AICAdboside. Iodotubercidin, an adenosine kinase inhibitor, suppressed the effect, implying that phosphorylation of AICAriboside by this enzyme was required. Further investigations revealed that AICAriboside displayed two major effects: (i) It decreased the release of 3H20 from [2-3H]glucose, synchronously with the inhibition of the production of lactate. This ,indicates inhibition of glucose transport or of an early step of glucose metabolism: phosphorylation into glucose-6-P (by hexokinase in FTO-2B cells), or isomerisation of glucose-6-P into fructose-6-P. (ii) It decreased the concentration of fructose-2,6-P 2, the main stimulator of phosphofructokinase (PFK)-I. This effect, which required at least 4 h of incubation, did not result from inactivation of PFK-2, but most probably from a decrease of fructose-6-P, due to inhibition of an early step of glucose metabolism. Fructose-l,6-P 2 decreased, most likely owing to the diminished stimulation of PFK-I.

In cell-free extracts of FTO-2B cells, phosphoglucose isomerase was 70% inhibited by 5 mM ZMP. Implication of this effect in the inhibition of glycolysis was, however, ruled out because residual activity remained 100-fold higher than that of hexokinase. Although no direct inhibition of hexokinase activity by ZMP could be evidenced as yet, it is tentatively concluded that A$CAriboside, by a still to be defined mechanism, primarily inhibits this enzyme. At longer time intervals, the inhibitory effect of AICAriboside on glycolysis is reinforced by the subsequent decrease of fructose-2,6-P2.

1 M.F. Vincent, F. Bontemps, G. Van den Berghe, Biochem. J. 281 (1992) 267 2 0 . Warburg, K. Posener, E. Negetein, Biochem. Z. 152 (1924) 309

Laboratory of Physiological Chemistry, International Institute of Cellular and Molecular Pathology, UCL 7539, Avenue Hippocrate 75, B-1200 Brussels, Belgium.

HIGH DOSE 6-MERCAPTOPURINE INFUSIONS IN CHILDREN WITH LYMPHOMA

CW Keuzenkams-Jansen RA De Abrea JPM B6kkerink MAH vd Heiiden

The mode of action of 6-mercaptopurine (6MP) in vitro is based on incorporation into DNA and RNA as thioguanine nuclcotides and inhibition of the purine de hove synthesis by methylthioinosinic monophosphate (MetlMP). We studied the metabolic routes of 6MP in 6 lymphoma patients receiving 24 hours infusions of 6MP. Pharmacokinetics, intracellular biochemistry and pharmacology were studied in plasma, erythrocytes and mononaclear cells. Blood samples were collected at 0, 4, 20, 24, 28 and 48 hours after start of the 6MP infusion (100 rag/m: in 0.5 hr followed by 1200 mg/m 2 in 23.5 hr). 6MP, its metabolites, ribonucleotides, nuclcosidas and bases were measured by HPLC. .: In plasma steady state levels of 6MP were reached soon after administration of 6MP in a concentration range of (~50 FM. Methylmercaptopurine (MeMP) appeared at t=4 (0.3-3 p.M) and the concentration decreased rapidly, like 6MP, after the end of the infusion. Methylmercaptopurineriboside (MeMPR) appeared at t=20 and remained at a steady state level (0-0.4 /~M) from t=24 till t=48. Two patients had a tumor lysis syndrome before start of the 6MP treatment. They demonstrated extremely high 6MP levels (35-60 ~.M) and longer 6MP half-life times. Relatively high MeMP levels (2-3 #M) and no MeMPR could be detected in these patients. Only these two patients had measurable amounts of thioxan- thine at extremely high levels (10-40/~M). MetlMP in erythrocytes was detected at t=4 and reached, like MeMPR, a steady state level (15-2000 pmol/10 m cells) at t=24. MetlMP in mononuclear cells (2-20 pmol/l(P ceils) was detected from t=20. The patients with a tumor lysis syndrome reached lower MatlMP levels as compared to the other children. Intraeellular thioguanine nuclcotides could not be detected in any of the patients, probably because these metabolites are rapidly incorparated into DNA. These data show large differences in 6MP metabolism in lymphoma patients. Patients without tumor lysis are probably able to incorporate 6MP into DNA and RNA and their blood cells contain MetlMP for more than 24 hours. In plasma of these patients no thioxanthine and less MeMP was detected. So, the anabolic route of 6MP in the cells of these patients is high. In contrast, in patients with a tumor lysis syndrome the catabolic route of 6MP is predominant: high levels of thioxanthine and relatively large amounts of MeMP. Our hypothesis for the predominant catabolic route of 6MP in tumor lysis patients is based on an excess of free nuclcosides and bases, derived from the lysis of tumor cells. The purine nuclcosides and bases may then compete with 6MP for the enzyme hypoxanthine- guanine phosphoribosyltransferase. This hypothesis is supported by the finding of high plasma levels of hypoxanthine (6 and 85 #M) and xanthine (25 and 350/~M) in these two patients.

Center for Pediatric Oncology, University Hospital, P.O.B. 9101, 6500 HB Nijmegen, The Netherlands. This project is supported by the DutchCancer Society (NUKC-92-79).

Volume 15 N[ 4 IQo~ FC~

HISTOCHEMICALAND CYTOCHEMICALDEMONSTRARqON OF THE MOLECULAR DEFECT IN TWO MUTANT FORMS OF HPRT DIHYDROOROTATE DEHYDROGENASE AND OXIDASE

M. L6mer. C. Becker. E. We~erle

In the present s tudy the enzyme dihydroorotate:ubiquinone o~dore- ductase (DHO DH] was invest igated in t issue samples of ra t and man, and in a mouse m a m m a carcinoma cell llne. Since the rnitochondrial DHO DH is connected to the functional respira- tory cha in 1, the enzyme activity could beeva lua ted tn s/ tu by channel - l ing the redox equivalents to artificial electron acceptors after cyanide- blocking of the chain at the stage of cytochrome oxidase. The principle of the techniques used for the detection of the dehydroge- nase activity was to employ a colourless, soluble tctrazolium sa l t which is reduced to a deeply coloured, insoluble formazan product. This could be detected by light microscopy, and, in a first approach, by flow cyto- metry if a f luorescent formazan was produced. The specificity of the dihydroorotate-dependent formazan production was confn'med us ing enzyme-inhibit ing drugs of high potency. The activity-pattern of DHO DH was comparable to tha t of succinate dehydrogenase, a marker of in- ner mitochondrial membrane. High activities were found in t i ssues with known t ranspor t /excret lon/regenerat ion/prol i fera t lve activities. The addit ional oxidase activity of the DHO DH, which was measured with the purified enzyme 2, was demons t r a t ed /n s/tu by employing the cer ium/diaminobenzld ine method for t rapping the hydrogen peroxlde resul t ing from direct reaction with oxygen. An u n u s u a l high o~ddase activity could be detected in the cardiac muscle by l ight microscopy. This was verified by u l t ras t ruc tura l s tudies which localized a cerium precipitate of h igh electron densi ty a t the inner mitochondrial membrane.

I M. LSffler, Cell Prolif. 25 (1992), 169. 2 G, Lakaschus and M. L6fller. Biochem. Pharmacol. 43 (1992}. 1025.

We thank C. Schalk. Institute of Molecular and Tumor Biology, University of Marburg for FACS analyses, and S. Angerm1~dler, Institute of Anatomy, Uni- versity of Heidelberg for electron microscopy. Financial support by the Sander-Stlftung and the Kulemann-Stiftung is grate- fully acknowledged-.

Ins t i tu t for Physiologische Chemie, Klinikum der Philipps-Universitftt Marburg, Karl-von-Frisch-Str., D-35011 Marburg, Germany.

A.M. Marinaki. E.H. Harley.

HPRTcaDe Town is a partially defective form of HPRT in which the enzyme demonstrates the unusual property of having acquired substrate inhibition by its purine base substrates I. Following PCR amplification of reverse transcribed /RRNA, products were directly sequenced and showed a A502G transition resulting in a change in Asp 135 to Asn. The latter is located in the putative PRPP binding domain and is conserved in all phosphoribosyl- transferases. This mutation requires re-interpretation of HPRT active site kinetics.

HPRTRo_dboschn e from a Lesch-Nyhan. . patient shows virtually no actlvlty. PCR ampliflcatlon of cDNA yielded a smaller product than normal due to a 47 base pair deletion corresponding to exon 7.

1 L.M. Steyn and E.H. Harley. J. Biol. Chem., 259 (1984) 338.

Dept of Chemical Pathology, University of Cape Town, Observatory 7925, South Africa.

ON THE ROLE OF THE IONIC COMPOSITION OF THE CULTURE MEDIUM FOR THE ROLE OF CELLULAR UPTAKE OF PHOSPHATE AND FOR THE RATE OF PHOSPHORYLATION OF ADDED PURINE

RIBOSIDE TO THE CORRESPONDING TRIPHOSPHATE

IV[. Marcussen, El Overgaard-Hausen and H. Klenow

Under a number of conditions large amounts of the t r iphosphates of added adenosine or analogs of adenosine may accumulate in Ehr l ich asci tes tumor cells. Almost a l l of the phosphate present in these t r iphosphates s t em from the extracel lu lar or thophosphate (P.~ and the i r r a te of accumulat ion may, therefore, in par t , depend on the ra te of up take of Pi. In med ia wi th h igh Na-concentrat ion l ike Ringer HEPES buffer and a t low pH (e.g. 6.5) the intraceUular concentrat ion of Pi (about 10 raM) increases by a factor of only about 2 wi th increas ing extracelhi lar Pi (up to 45 re_M). Never thaless the presence of pur ina riboside (nebular/he) may cause not only a drast ic decrease in intraceUular Pi bu t also a rapid up take of ext race l lu lar Pi resu l t ing in accumulat ion of la rge amounts of pur ina r iboside t r lphosphate . The l inear increase in pur ine r iboslde t r iphosphate may in about 2 h resu l t in a to ta l content of low ~nolecular phosphate compounds t h a t is about 10-fold t h a t of cells not t rea ted wi th purine riboside. I t t hus appears t h a t the in t racel lu lar phosphorylat iou of pur ine r iboside faci l i tates a grea t ly increased net up take of Pv In media-buffers w i th a h igh K-concentration increas ing concentration of extraceUular Pi gives rise to an unexpected high rate of accumulation of intracellular Pi. Under such conditions, or at high pH values (e.g. 7,9), the total Pi uptake was almost unaffected by the presence of purine ribosids even though considerable amounts of it were converted to the triphosphate.

Dept. of Medical Biochemistry and Genetics, Laboratory of Medical Biochemistry B, The Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.

FAMILIAL JUVENILE HYPERURICAEMIC NEPHROPATHY IS ALSO A DISEASE OF CHILDHOOD.

M.B. McBride. H.A. Simmonds, F. Moro, J.S. Cameron, C.S. Oqg, D.G. Williams, S. Riqden and G. Haycock.

Familial juvenile hyperuricaemic nephropathy (FJHN) is a dominant condition with high penetranee which affects a group of young men and women who develop early progressive renal dysfunction. FJHN has often been considered 'familial nephritis' until an isolated attack of gout (rare in renal failure) has highlighted the underlying disorder. Gout and hypertension are inconsistent features. The genetic basis remains unknown.

FJHH has, hitherto, been considered rare. Detailed studies of 88 cases from 14 kindreds have been reported from 7 countries, excluding the UK, 36 of these cases have been children. We have now studied 90 individuals from 34 kindreds in which the index case presented with gout and found that the hallmark of the disease is hyperuricaemia associated with a low clearance of uric acid relative to creatinine (FEur), disproportionate to age, sex and degree of renal failure. On this criteria we identified 65 affected individuals, 25 of whom were reputedly healthy. The study included 27 children (one a female seen with gout at 9 years). A reduced FEur has been found in 18 of the children, including 5 with as yet normal renal function, suggesting that renal urate hypoexcretion precedes a fall in the GFR.

Our findings indicate that FJHN is also a disease of childhood. They underline the importance of detailed family studies in all kindred members. This is essential since, in our experience; a) allopurinol appears to have ameliorated the progression of the renal damage in patients treated for up to 25 years; b) a successful prognosis appears to depend on the renal function at diagnosis and the absence of hypertension, or its adequate control.

Purine Research Laboratory, Renal and Paediatric Nephrology Units, UMDS Guy's Hospital, London, UK.

~ ] 0 l'h,lr.m,y H~,tld ~ ,q, wmc

INCREASED RENAL VASCULAR RESISTANCE IN FAMILIAL NEPROPATHY ASSOCIATED WITH HYPERURICEMIA OR GOUT.

ME Miranda. JG Puin FA Mateos. JO V~zouez.

We determined the renal plasma flow (RPF), renal vascular resistance (RVR) and filtration fraction (FF) in 11 subjects belonging to 3 families with familial nephropathy associated with hyperuricemia or gout (McKusik, 162000). Ten patients showed hyperuricemia, 5 had gout, 8 decreased glomerular filtration rate (GFR) and 5 arterial hypertension. All the 11 patients showed a decreased RPF (267+110 mL/min/1.73 m 2 [reference, 709+30]), increased RVR (28.0_+16 mmHg/mL/ l .73 m2 [reference, 7.6_+ 0.6]) and normal FF(22.5_+3.1% [reference, 20.7+1.2]). There was a significant correlation between RPF and 24 hour urinary urate excretion (r=0.74, P<0.01). A decreased RPF and increased RVR was present in one patient wih normal GFR, blood pressure and serum uric acid levels. Another patient showed hyperuricemia, decreased RPF and increased RVR as the only manifestations of the disease. After 4 years of follow-up his GFR decreased by 30% despite an adequate contro~ of hyperuricemia. In summary, patients with familial nephropathy associated with hyperuricemia or gout show a decreased RPF and increased RVR even in the absence of other manifestations of the disease. The observed correlation between RPF and uric acid excretion suggests that altered renal hemodynamics may account for the urate underexcretion, hyperuricemia and the entire clinical picture of this familial syndrome.

Dpt of Internal Medicine and Clinical Pharmacology. La Paz University Hospital. Paseo de la Castellana, 261. 28046 Madrid, Spain.

ADENYLOSUCCINATE LYASE DEFICIENCY: A CASE REPORT

C. Salerno, A. Lomonte, O. Giardini, C. Crifo'

Adenylosuccinate lyase deficiency is a recently described inborn error of purine metabolism discovered in Benelux countries [i] and diagnosed hitherto in 12 children, belonging to 4 nationalities (Belgian, Dutch, Moroccan and Turkish) [2]. Affected children are normal at birth, but psychomotor retardation and autistic features often become evident in the first two years.

We have found a new case of adenylosuccinate lyase deficiency in the course of a systematic screening of urines of more than 100 patients with severe psychomotor retardation belonging to the Italian Association of Parents of Autistic Subjects. HPCL detection of succinyladenosine (S-Ado) and succinylaminoimidazole carboxamide riboside (SAICA-R) was the technique of choice for the screening.

The affected girl (now 9 years old) is the second daughter of unrelated Italian parents. A few months after birth, frequent crying attacks, motor restlessness and hypertonicity were noticed. Eye contact was difficult, while reaction to auditory stimuli was exaggerated. Psichomotor development was delayed (she was standing alone at 15 months and walking after 2 years; up to now, she speaks unintelligibly in short sentences with an incorrect structure and seems to understand very little of what is said to them). At age of 5 years, generalized tonic-clonic convulsions were noted for the first time. There is an intermittent external strabism with crisis of convergence spasm. Striking bouts of extreme agitation, especially involving the arms and legs, are common. Growth and head circumference are normal.

The patient excretes approximately 130 nmol S-Ado / min in the urine, while [S-Ado] in plasma is about 3 uM. The [S-Ado] to [SAICA-R] ratio is about 1.2. Serum urate concentration is 2-4 mg/dl. Intravenous injection of 200 mg / kg fructose causes a decrease of serum urate concentration of about 1 mg/dl and a slight increase of serum [Mg]. Erythrocyte adenylosuccinate lyase appears to be unstable with time.

i. J. Jaeken & G. Van den Berghe, Lancet 2 (1984) 1058. 2. F. Van den Bergh, PhD Thesis, univ. Louvain, 1992, 45.

Dept. of Human Bio~athology, Dept. of Biochemical Sciences and Inst. of Paedriatrics, University of Roma La Sapienza, 00161 Roma, Italy

2',2'-DIFLUORO-DEOXYCYTIDINE INCORPORATES INTO RNA AND DNA AND INHIBITS THEIR SYNTHESIS.

V.W.T. Ruiz van Haperen, G. Veerman, J.B. Vermorken, G.J. Peters.

2',2'-difluoro-deoxycytidine (Gemcitabine, dFdC) is a deoxycytidine analogue with established anti-tumour activity in the clinic, dFdC is assumed to exert its antitumour effect through incorporation into DNA. Since the RNA-directed effects were not clearly defined, we studied the incorporation of dl:dC into DNA and RNA and its effect on nucteic acid synthesis. We determined incorporation of dFdC into nucleic acids of A2780, a human ovarian carcinoma cell line, Colon 26-10, a mudne colon carcinoma cell line and in CCRF-CEM, a human leukemic cell line. For this purpose we used two different methods; an acid precipitation assay and a CsCI density gradient, after exposing the cells for 4 or 24 h to 0.1 or 1 I~M dFdC. Synthesis of DNA was determined by [2-14C]-thymidine (14C-TdR) incorporation, RNA synthesis by [5- 3H]-uridine (3H-UR) incorporation. Incorporation of dFdC into DNA was time and concentration dependent. A2780, the most sensitive to dFdC, incorporated most dFdC (3-'7 fold compared to C26- 10). We also observed incorporation into RNA, which was only concentration dependent, within the time period used. The solid tumour cell lines incorporated much more dFdC into RNA (6-14 fold) than the leukemic cell line, especially at 1 I~M dFdC. dFdC incorporation into RNA was confirmed by separation of RNA and DNA using CsCI gradient centrifugation. The same pattern was observed as w~th the acid precipitaion assay, while contamination of RNA by DNA was less than 0.4%. As to the synthesis of the nucleic acids, in all three cell lines dFdC inhibited DNA synthesis completely, at 1 I~M after 24 h exposure. RNA synthesis could be inhibited completely only in CCRF-CEM ceils, after 24 h exposure to 1 p.M of dFdC. In the two solid tumeur cell lines RNA synthesis was also inhibited, however to a maximum of 60%. In conclusion, dFdC interferes with both DNA and RNA synthesis. The latter effect has not been recognized before and it has to be determined wether it is related to either anti-tumour activity or toxicity.

Dept. of Oncology, Free University Hospital, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands.

DEPRESSED PURINE CATABOLITE PRODUCTION IN THE POSTISCHEMIC HEAR3~

A 31p-NMR ASSESSMENT OF CYTOSOLIC METABOLITES.

Smolenski, M.H. Yacoub. A.M-L. Seymour,

Brief period of ischemia causes increase in adenosine and total purine catabolite release from the heart. However, if ischemia of similar duration is repeated, this increase in purine catabolite release is markedly reduced. The mechanism of this phenomenon is unclear. In this study an isovolum- ic Langendorff perfused rat heart model was used. Hearts were subjected to (A) 1 min ischemia at 40 rain of perfusion, 10 rain ischemia at 50 rain of perfusion and 1 rain ischemia at 85 rain of perfusion or to (B) 3x I min ischemia at 40 rain, 60 rain, and 85 min of perfusion. ~ntracellular concentrations of ATP, phosphocreatine (PCr) and intracellular inorganic phosphate (P) were assessed using 3~p nuclear magnetic resonance spectroscopy (NMR). Purine catabolite concentrat ion in the coronary effluent was determined by HPLC. Mechanical function of the heart (left ventricular developed pressure - LVDP) was determined using a balloon inserted into the left ventricle. Both in (A) and (B) P. increased two fold during 1 min ischemia at 40 min of perfumon. PCr decreased concomitantly by 30% but no change n ATP was seen. Prior 1 min ischemia at 85 rain of perfusion, ATP concentra- tion was 65%, Pi was 60% and PCr 180% of the initial values in group (A) whilst these concentrations remained unchanged relative to starting levels in group (B). During 1 rain ischemia at 85 rain, concentrations of P. and PCr reached the same levels as during the first ischemic incident both in group (A) and (B). Relative change in comparison to values ,~nmediately before was thus much more pronounced in group (A). Purina catabotite release increased by 0.5/JM immediately after 1 rain ischemia at 40 min, but only by 0.05 pM at 85 min in group (A). These increases in purine release remained unchanged in group (B) during 1 rain ischemia at 40, 60 and 85 rain. Contractile function (LVDP) recovered to value before 10 rain ischemia in group (A) and was maintained constant in group (B). In summary, despite ten fold reduction in purine catabolite release, myo- cardial concentrations of P. and PCr during 1 rain ischemia before and after 10 rain ischemia wer~ similar and lower was only ATP concentra- tion during the second 1 rain ischemic event. However, PCr level was higher and P. concentration lower prior to second 1 rain ischemic inci- dent. Conse~luently, decreased nucleotide catabolite production in our experimental model seems to be related to reduction of nucleotide pool size and/or increased phosphorylation status before ischemia, but in- volvement of other factors still can not be excluded.

M.R.S. Group, Cardiothoracic Surgery, National Heart and Lung Institute at Harefield Hospital, Harefield, Middx UB9 6JH, U.K.

Volume 15 Nr 4 199]

MUTATIONAL LOSS OF DEOXYCYTIDINE KINASE (dCk) ACTIVITY AS A CAUSE OF ACQUIRED 1-S-D- ARABINOFURANOSYLCYTOSINE- (AraC) AND 5 -AZA-2'- DEOXYCYTIDINE- (AzadC) RESISTANCE IN ACUTE MYELOID LEUKEMIA (AML).

A.P.A+ Steqmann. M.W. Honders, R. Willemze. J.E. Landeqent.

Acquired drug resistance to the dC-analogues AraC and AzadC in the treatment of AML, one of the major obstacles for longterm beneficial therapy, is often associated with deficiency of the pyrimidine nucleoside kinase dCk. Recently, in human T-cell lines this lack of enzymatic activity was shown to be associated with mutations in the dCk-gene, leading to severely reduced levels of the enzyme. We used cell lines derived from an in vivo rat model for AML to study mutational loss of dCk activity and the mechanisms underlying these mutations. One cell line (RCL/0) is sensitive to the drugs and has a normal dCk activity, a second (RCL/A) displays AraC- and AzadC-resistance that was induced ex vivo in the animal model; it shows no dCk-activity for AraC and AzadC, as well as a severely impaired enzyme activity for the metabolization of dC. We exposed RCL/O cells to gradually increasing concentrations of either AraC or AzadC and thus generated two independant AraC- and AzadC-resistant cell lines (RCL/0-A and RCL/D resp.). Using RT-PCR-techniques with oligonucleotide primers chosen from the human dCk coding region , all AraC-resistant cell lines were shown to express dCk-specific sequences with aberant restriction sites as compared to amplicons generated from the (normal) dCk-mRNA coding region of RCL/0. Southern blot analysis of RCL/A and RCL/0- A genomic DNA revealed a genomic rearrangement of the dCk gene. In the AzadC-resistant RCL/D no aberrant PCR-amplicons or Southern hybridization patterns were detected. SSCP analysis however suggests a point mutation in the RCL/D dCk-gene. The data suggest distinct mutational events, underlying AraC- and AzadC-resistance respectively, in this model.

DETERMINATION OF PHOSPHORIBOSYLPYROPHOSPHATE SYNTHETASE ACTIVITY. A NEW SIMPLIFIED METHOD.

R.J. Torres. F.A. Mateos. J.G. Pui~, Phosphorlbosytpyrophosphate synthetase (PRPPs) superactivity is an inherited

inborn error of metabolism which causes gout with over-excretion of uric acid associated, in some instances, with neurodevelopmental impairment. Screening for defects in PRPPs activity is complicated by the variety of kinetic alterations detected among affected families, being the most common the catalytic and the regulatory defeCts. Currently employed methods for measurement of PRPPs activity are cumbersome and expensive, requiring an auxiliary enzyme reaction, a radioactive nucleobase, and multiple incubations. The aim of the present study is to describe a simplified, single step, nonisotopic method for determination of PRPPs in hemolysate.

Charcoal-treated hemolysate is incubated in a pH 7.4 reaction mixture containing: 50 mM TfisC1H, 5 mM MgC12, 1 mM EDTA, 0.4 mM DTT, 0.5 mM ATP, 0.35 mM Ribose 5-phosphate, 32 mM NaH2PO4 and 0.25 mM PI-P5 flladenosin 5' pentaphosphate (Ap5A, an inhibitor of the adanylate kinase). The reaction is stopped by addition of 0.1 M EDTA, and the samples are centrifuged in Amicon cones. The resulting filtrate is injected into an HPLC ,aBondapak C18 column and eluted with 0.2 M KH2PO4, pH 6, at 1.3 ml/min. Absorbance is measured at 254 nm, and PRPPs activity is expressed as nmol of AMP generated/h/ mg hemoglobin.

Adenylate kinase activity is fully inhibited by Ap5A, allowing the accurate determination of AMP. The method is sensitive and reproducible, being PRPPs activity linear with the amount of added hemolysate and with time of incubation, and mean and variance values (91.9 _+ 17.8 nmollh/mg) compare closely with those reported using other, more complicated, assay procedures (1). There is a significant correlation between the PRPPs activity measured by this one step assay and the PRPPs activity determined by a modification of the two step assay employing adenine phosphoribosyltransferase as coupled enzyme (r=0.993, p<0.001). The hyperbolic curve relating Pi concentration to initial reaction velocity is shifted to sigmoidal by addition of 0.02 mM GDP whioh inhibite PRPPs activities only at Pi concentrations < 2 raM. This suggezt that this method should provide sensitive and accurate screening for regulatory as well as catalytic defects underlying PRPPs superactivity.

We conclude that the new method presented has important adventages over the currently employed methods, and may provide a valuable diagnostic tool for a better understanding of the metabolic basis of uric acid overproduction and gout.

I. Losman, et al. J Lab Clin Med 1984; 103: 932.

Clinical Biochemistry and Internal Medicine Sections. La Paz University Hospital. Paseo de la Castellana n" 261. 280,16 Madrid. Spain.

THE IMPORTANCE OF THE METHYLATION ROUTE FOR 6- MERCAPTOPURINE CYTOTOXICITY IN MOLT F4 HUMAN LYMPHOBLASTS.

E,H, Stet+ R.A. O~ Abreu. G.M. Vo~els-MenLi0k. L .HJ . Lamboov. J.P+M. B~kkerink. J.M.F. Tri)bels. 6-Mercaptopurine (6MP) is used in the maintenance treatment of children with acute lymphoblastic leukemia. Extensive intranellular metabolism of 6MP is required for its oytotoxicity. First, 6MP is converted into thin-IMP (tIMP) by the enzyme hypoxanthine guanine phosphoribosyltransferase, tIMP is a branch-point in 6MP metabolism. The first route of tIMP metabolism is conversion into thioguanine nocleotides by enzymes of the purine interconversion pathway. In this route IMP dehydrogenase (IMPDH) is the rate-limiting enzyme. Thioguanine nuclenddes are incorporate~l into DNA of the cells, inducing delayed cytotoxicity. In general it is accepted that this is the main caus~ for 6MP cytotoxicity. The second route for tIMP metabolism is methylation into methyl-thio-IMP (Mo- dMP). This reaction is catalyzed by the S-adenosylmethionine (SAM) dependent enzyme thiopurine methyltransfera.se in a conversion by which S-adenosyl-L- homocysteine is formed. Me-tIMP is a strong inhibitor of the purine de novo synthesis. Inhibition of this pathway leads to a depletion of porine nucleotides, and induces inhibition of DNA synthesis. This metabolic route may also contribute to cellular cytotoxieity. In this study the importance of the methylation route for 6MP cytotoxicity was studied in Molt F4 human malignant lymphoblasts. Cells were treated with 6MP combined with mycophenolic acid, an inhibitor of IMPDH, and therefore an inhibitor of formation of thioguanine nucleotides. As a result of addition of 0.5 #M MPA to treatment with 2 .uM 6MP, less thio-GMP and more Me-tIMP were formed. Under these conditions cytotoxicity was increased as compared to 6MP alone. As a result of addition of AICAR, an intermediate of the purine de novo synthesis to the treatment with 6MP and MPA, cytotoxicity resembled that of MPA alone. We therefore concluded that methylation is important for 6MP cytotoxicity. Furthermore, we determined the effects of 6MP on the SAM and the SAH concentradons in Molt F4 cells. Treatment with 6MP decreased SAM, and increased SAH COncentration as compared to untreated ceils. SAM is an important methyl-donor in eukariotic cells, and is involved in methylation of DNA, RNA, proteins and phospholipids. So, any effect on the SAM/SAH ratio may exert effects on these methylation reactions. Therefore, imbalance of the SAM/SAH ratio may he an other important mechanism by which 6MP induces deregulation of cellular processes, which may ultimately result in cellular cytotoxicity.

Center for Pediatric Oncology SE Netherlands, University Hospital Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. This project is supported by the Dutch Cancer Society (NUKC 89-13)

I~GULATION OF ~ BIFUNCTIONAL CYTOSOLIC 5'-NUCLEOTIDASFJNUCLEOSIDE PHOSPHOTRANSFERASE

*M,G. Tozzi. R Pes], M Turriani,S. Allegrini. M. CamicL P.L I o a ~

Cytosolic 5'-anclentidase, an ubiquitous enzyme which preferentially hydrolyzes 6- hydroxypufine nucleoside monophosphate% is regulated by energy charge, 2,3- diphosphogiycerate and phosphate (1-3). The enzyme appears to be present at higher concentration in cells and organs with high nucleic acid turnover (4). Cytosolic 5'- nuclcotidase behaves as a bifunctional enzyme since it catalyzes the transfer of phosphate from a nucleoslde monophosphate to a nucleoside aceeptor, probably forming an enzyme-phosphate intermediate, thus operating a mononucleotide int~annversion (5). In the absence of a suitable nuchioside, the enzyme-phosphate complex is hydroiytically cleaved. The understanding of the regulation of the two activities of this enzyme is depending on the possibility to measure the rate of formation of the three reaction products: phosphate, nuclenside, and mononucleotide, in the same assay condition. Cytosolic 5' nucleotidase, has been purified to electrophoretic homogeneity from calf thymus. Its characteristics resulted to be identical to those of the enzyme purified from human colon carcinoma (2).We developed analytical methods utilizing HPLC and Capillary Electrophoresis to follow both enzyme activities as a fimction of pH and energy charge variation, as well as nudeoside acceptor and nucleotide donor concentrations. Our preliminary results indicate that the two activities show different optimum pH. Furthermore both activities are increased in the presence of high energy charge, hut they display different ratios. In fact, 5'-nucleotidase is the main activity at low energy charge and slightly acidic pit, while phosphotransferase is favoured at high energy charge and pH around the neutrality. The study of the regulation of the enzyme is of great importance for the understanding both of its physiological role, and of its role in the "activation" or "inactivation" of purine prodmgs.

1. Zimmermann H. Biochem. J. 385 (1993) 344. 2. Tozzi M.G. et al. Arch. Biochem. Biophys. 291 (1991) 212. 3. Itoh l~ et al. Biochem. J. 235 0956) 847. 4. Itoh R. and Yamada K. Int. J. Biochem. 23 (199l). 5. Worku Y. and Newby A.C. Biochem. J. 205 (1982) 503.

Dipartimento di Fisinlogia e Biochimica, Universlt~i di Pisa, via S. Maria 55, 56100 Pisa Italy *Istituto di Chimica Biologica, Universitl di Sassari, via Muroni 22/A, 07100 Sassari, Italy

F]2 +"+',,.,..,r Jl;,,l,l +~: +'q+,mr

EIq'HANCED EXPRESSION OF THE CTP-SYNTHETASE GENE AFTER DEPLETION OF ITS END PRODUCT CTP

A.A. van den Berg, J.R. Meinsma, H. van Lenthe, A.H. van Gennip and A.B.P. van Kuilenburg.

The activity of the enzyme CTP-synthetase in human leuke- mic cells is increased in a transformation associated manner, compared to that observed in normal human blood cells. Therefore, inhibitors of CTP-synthetase have been proposed to be good candidate anti-leukemic drugs. The relationship between the expression of the CTP-synthetase gene and the size of the CTP pool in human cells is not known. However, this relationship might be very important in the light of the sensitivity of cells for inhibitors of CTP-synthetase. Therefore, we studied this relation- ship in HL-60 human promyelocytic cell-line cells.

HL-60 cells were incubated with 25 MM of 3'-deazauridine (DAU), with i00 ~M of cytidine (Cyd) or 1.25 % DMSO. Initially, this led to a progressive depletion of the CTP pool (DAU), an increased CTP pool (Cyd) or an unchanged CTP pool (DMSO). RNA was purified from all HL-60 cells. The expression of the CTP-synthetase gene at the level of its mRNA was determined after hybridisation with radio- labeled DNA probes of the CTP-synthetsae gene. During incubation with DAU, the amount of CTP-synthetase mRNA was increased in CTP depleted cells after 2 to 24 hours. Beyond 24 hours, the mRNA encoding the CTP-synthetase was decreased and finally became undetectable. Also, in the presence of DMSO, the mRNA for CTP-synthatse diappeared within 48 hours. Because the HL-60 cells differentiated into functional neutrophilic cells, both after treatment with DAU and treatment with DMSO, it might be that the down-regulation of the CTP-synthatse gene is directly related to the maturation process. In cells incubated with Cyd, the changes that Occurred in the amounts of CTP-synthetase mRNA are being studied now. So far, we conclude that in HL-60 cells the expression of the CTP- synthetase gene, at the level of the mRNA transcript, is up-regulated after depletion of the intracellular CTP pools.(Supported by the Dutch Federation for Pediatric Oncology Research, Grant SKK 89-01).

Academic Me4ical Center, Deps. Pediatrisc Oncology and Clinical Chemistry, F-0-105, Meibergdreef 9/ 1105 AZ, Amsterdam, The Netherlands.

INTERFERON MODULATION OF 5-FLUOROURACIL IN COLON TUMOR CELL LINES AT DIFFERENT FOLATE LEVELS

C.L van der Wilt, P. Noordhuis, K. Smid, H.M. Pinedo, G.J. Peters

The relatively low response rate of standard 5-fluorouracil (FU) therapy ~n advanced oolorsctal cancer can be increased by biochemical modulation at different levels. Leucovorin (LV) modulates by increasing the reduced folate pools, which might be limiting for maximal inhibition of thymidylate synthase ('iS), mediated by the FU metabolite FdUMP. The mechanism by which interferons (IFN-a and IFN-7) modulate FU activity are not completely elucidated, but IFN-~ may increase FdUMP pools in the cell, thereby increasing FdUMP bindng to TS and TS inhibition, while IFN-7 has shown an effect at the level of TS expression. We used two colon cancer cell lines (C26-10, murine and WiDr, human) to study the modulating effects of LV and IFNs. In these cell lines the antiproliferative effects of FU and FUdR, a FU derivative which is 'a more potent inhibitor of TS, could not be modulated with LV (10 ~.M). The folates in the culture medium (2.3 I~M) might be sufficient to achieve maximal TS inhibition. Sublines of the colon celt lines (C26-10/F and WiDr/F) were adapted to physiological levels of folates and maintained at 0.25 nM folinic acid. Sensitivity for FU and FUdR remained unchanged but addition of LV (10 #M) increased FUdR mediated growth inhibition 2-fold in C26-10/F. Activity of TS was increased in C26-10/F (1.3x) and WiDr/F (2.3x) compared to the parental lines. Neither in the parental C26-10 nor in the subline C26-10/F IFN-~z or IFN-7 (100 U/ml) of human origine could enhance FU antitumor effect. IFN-7 was an active modulator in WiDr and WiDr/F and a 2-fold increase of FU activity was observed. In contrast to WiDr, WiDr/F also showed sensitivity to IFN-~ modulation (about 2-fold increase of FU activity), while IFN-r and IFN-y in combination more than additively enhanced FU activity. Addition of thymidine (TdR) in these growth inhibition tests reversed the antiproliferative activity of FU and FUdR alone and that of combinations of these drugs with LV. The antiproliferative effect of high dose single IFN-~ or IFN-7 could not be reversed by TdR. TS activity measured in intact cells by incorporation of 3H-deoxyuridine into DNA was inhibited by FU in all cell lines. IFNs did not influence the extent of 3H- deoxyuridine incorporation, but enhanced TdR incorporation. These experiments underline the importance of folate levels in the modulation of FU activity. At physiological folate levels LV was useful as a modulator that acted at the level of TS inhibition, since TdR reversed the growth inhibitory effects. Although IFNs were more active at physiological folate levels, the inhibition of TS was not directly modulated by IFNs.

Free University Hospital, dept. Oncology, P.O. BOX 7057, 1007 MB Amsterdam, The Netherlands

ALTERNATIVE SPLICING EVENTS IN THE 5'-END OF THE HUMAN AMPD2 PRIMARY TRANSCRIPT GENERATE N-TERMINAL

VARIANTS OF ISOFORM L.

F. Van den Berah. R.L Sabina

Higher eukaryotes express multiple isoforms of AMP deaminase (EC 3.5.4.6.). In humans, at least three variants termed M(uscle), L(iver) and E(rythrocytes) have been characterized. Previous molecular studies have reported three genes: AMPD1 (1), AMPD2 (2) and AMPD3 (3) that produce transcripts encoding tsoforms M, L and E respectively. Documented alternative splicing events in the 5'-ends of the AMPD1 and AMPD3 primary transcripts are predicted to generate isoform M and E variants, respectively, differing at or near their N-terminii.

Multiple human tissue Northern blot analysis (2) has demonstrated an approximately 4 kb AMPD2.transcript in non-muscle tissues. These results indicate a lack of several hundred basepairs from the 5'-end of existing cDNA sequence. Additionally, a second smaller and more abundant AMPD2 transcript appears to be expressed predominantly, if not exclusively, in the brain. Our aims were to identify the extreme 5'-end of the AMPD2 gene, and to investigate suspected alternative splicing events in ffs primary transcript.

A human cerebellum kgtl0 cDNA library was used to isolate additional AMPD2 clones using, as a probe the previously described 5' human T- cell lymphoblast clone: Hut6A (2). Sixteen positive plaques were isolated and the recombinant inserts from twelve were subcloned and sequenced. Nucleotide sequence alignments between these and existing human AMPD2 cDNA clones has demonstrated three divergent extreme 5'-ends, which contribute at least 500 bp to the known sequence. Accordingly, a human AMPD2 genomic clone was used to identify three exons which contain the new 5'-end sequences. Subsequent RNase protection analyses were used to demonstrate both cassette and mutually exclusive alternative splicing events involving the three identified exons. At the predicted protein level, our results indicate at least two different extreme N-terminii. These data, therefore, infer an additional level of complexity to isoform L expression in human tissues and cells.

1. R.L. Sabina, et al. J. Biol. Chem., 265 (1990) 9423. 2. M.T. 8ausch-Jurken, et aL J. Biol. Chem., 267 (1992) 22407. 3. D.K. Mahnke-Zizelman, R.L. Sabina. J. Biol. Chem., 267 (1992) 20866.

Dept. of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee Wi 53226, USA.

COMPARISON OF DIHYDROPYRIMIDINE DEHYDROGENASE ACTIVITIES BETWEEN LEUKOCYTES AND FIBROBLASTS IN A FAMILY OF A PA- TIENT WITH THYMINE-URACILURIA

A.H. van GENNIP, H. van LENTHE, N.G.G.M. ABELING, H.D. BAKKER AND A.B.P. van KUILENBURG

Thymine-uraciluria can be the result of a deficiency of one of the enzymes: dihydropyrimidine dehydrogenase (DHPD, EC 1.3.1.2) or dihydropyrimidinase (DHP, EC 3.5.2.2). DHPD activity can be measured in lymphocytes, monocytes, fibroblasts and liver. In contrast, DHP ac- tivlty is not expressed in leukocytes or fibroblasts and therefore has to be measured in liver. As DHPD deficiency is a recently discovered disorder, it is important to investigate whether the defect is equally expressed in the tissues mentioned. We had the opportunity to study leukocytes and fibroblasts in the family of a patient with the defect.

Patient BRB, a boy aged 2 yr, was investigated for metabolic abnormalities because of psychomotor retardation and ocular abnormalities. He appeared to have thymine-uraciluria. It was decided to investigate also his parents and 2 brothers, although healthy, for this abnormality. For this purpose 24 h urine samples, blood and skin biopsies were collected from the patient and his family. In the patient thymine-uraciluria was confirmed, while the pyrimidine excretion profiles of the family members were normal.

The activity of DHPD was measured in lymnhocytes and cultured fibroblasts after incubation with 2-~C thymine by HPLC analysis and on-line radioactivity measurement of the reaction products. DHPD activities were undetectably low in the lymphocytes and fibroblasts from the patient. In one brother they were normal, while the other brother and the parents showed intermediate values. The results from the measurements in both tissues led to identical conclusions, giving support to the assumption that leuko- cytes as well as fibroblasts are appropriate for confir- mation of the diagnosis on the enzyme level.

Academic Medical Center (AMC), Divisions of Pediatrics and Cllnical Chemistry, Meibefgdreef 9, 1105 AZ Amster- dam, The Netherlands

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INHIBITION OF CTP SYNTHETASE INDUCES DIFFERENTIATION OF HL-60 CELLS AND DOWN-REGULATION OF THE C-MYC ONCOGENE.

A.B.P. Van Kuilenburs, A.A. Van den Bers. J.R. Meinsma. R.J. Slin-oerland and A.H. V~n Gennip.

CTP synthetase and IMP dehydrogenase are the two 'key-enzymes' for the de novo biosynthesis of CTP and GTP, respectively. Inhibition of IMP dehydrogenase depletes the inlxacellulur guanine nucleotide pools and induces granulocytic differentiation of HL-60 ceils. Furthermore, the induction of HL-60 differentiation to granulocytes is associated with specific changes in the protooncogene expression (1,2). These findings have suggested that guanine ribonucleotides play an important role in the regulation of the differentiation of leukemie-myeloid cells. So far, the role of CTP synthetase and cytosine nucleotides in the process of differentiation and regulation of oncogene expression in HL-60 cells has hardly been studied. Therefore, we studied the pattern of c-myc expression when HL-60 cells were induced to differentiate with either DMSO or with an inhibitor of CTP synthetase.

When HL-60 ceils were incubated with 25 I.tM 3'-deazauridine a rapid depletion of the CTP ribonucleotides was observed within 2 h which persisted at least until 24 h. The decline in the CTP levels reflects the inhibition of CTP synthetase by deazaUTP which is a competitive inhibitor of the enzyme. However, a small increase in the UTP pool was observed which can be explained by the fact that uridine and deazauridine are both metabolised by the same uridine/cytidine kinase. The progressive increase in the levels of ATP and GTP until 24 h, is most probably due to stimulation of the purine de novo pathway by PRPP which accumulates after inhibition of the pyrimidine de novo pathway.

The mRNA levels of the c-myc oncogene was followed up to 72 h of exposure of HL-60 ceils to either DMSO or 3'-deazauridine. Treatment of HL-60 ceils with DMSO resulted in a very rapid decline of the c-myc oncogene transcript and no c-myc mRNA could be detected after 2 h. Despite the very rapid loss of c-myc mRNA, the HL-60 cells continued to progress through one cell cycle. Therefore, there appeared to be no direct link between the c.myc gene expression and the onset of proliferation arrest when HL-60 cells were induced to differentiate with DMSO.

The inhibition of CTP synthetase by deazaUTP resulted in a progressive decline of the c-myc transcript to undetectable levels within 48 h. Since CTP synthetase has an important role in providing the necessary precursors for RNA and DNA biosynthesis, the observed decrease in proliferation rate might be due to the depletion of CTP nucleotides. The proportion of HL-60 cells capable of generating an oxidative burst, a phenomenon characteristic of mature granulocytic cells, increased to approximately 50% after 5 days.

Our results demonstrate that the inhibition of CTP synthetase is associated with the down-regulation of the c-rayc oncogene which is a prerequisite in the process of terminal differentiation of HL-60 cells.

1) S.M. Khurbanda et al. Cancer Res., 48 (1988) 5965. 2) L.S. Mitchell et al. Differentiation, 49 (1992) 119.

Academic Medical Centre, Divs. EKZIkinder AMC and Clinical Chemistry, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands.

PURINE NU~DE METABOLISM IN HGPRT DEFICIENT RAT NEUROMA CELL LINE

E__~.Zoref-Shani, Y_:_.BromberH, S__~.Brosh~ Y__~.Sidi, O_.~.Sperlin K

An HGPRT-deficient rat neuroma cell line (BI03 4C), was utilized as a model tissue in search for the biochemical basis of the neurological manifestations of the Lesch-Nyhan syndrome (LNS). The cells exhibited an almost complete absence of uptake of guanine and of hypoxanthine into intact cell nucleotides (0.92 % and 0.69 % of normal, respectively); a significant increase in the availability of PRPP; a 3 to 4 fold acceleration of the rate of nucleotide synthesis de novo; a normal excretion of xanthine, but 15 fold increase in the excretion of hypoxanthine into the culture media; a normal cellular purina nucleotide content (including the absence of Z-eucleotides), but slightly enhanced turnover of adenine nucleotides and an elevated [rl'Pcontent. The results suggest that under physiological conditions guanine salvage does not occur in the normal neurons but that hypoxanthine salvage is of great importance in the homeostasis of the adenine nucleotide pool. However, in the HGPRT- deficient neurons the lack of nucleotide salvage synthesis from hypoxanthine is adequately compensated by the enhanced de novo nucleotide synthesis. The results did not furnish evidence in support of the possibility that GTP or ATP depletion, or Z-nucleotide accumulation occur in HGPRT- deficient neurons, but point to the possibility that elevated hypoxanthine concentration in the brain ~nay have an etiological role in the pathogenesis of iNS.

Dept. of Chemical Pathology, Sackler School of Medicine, Tel Aviv University, Tel Aviv and Depts. of Clinical Biochemistry and of Medeicine D, Beilinson Medical Center, Petah-Tikva, Israel.

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