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The majority of human pancreatic adenocarcinomas display a ductal phenotype; experimental studies indicate that tumors with this phenotype can arise from both acinar and ductal cells. In normal pancreas acinar cells, the pancreas transcription factor 1 transcriptional complex is required for gene expression. Pancreas transcription factor 1 is a heterooligomer of pancreas-specific (p48) and ubiquitous (p75/E2A and p64/HEB) basic helix-loop-helix proteins. We have examined the role of p48 in the phenotype of azaserine-induced rat DSL6 tumors and cancers of the human exocrine pancreas. Serially transplanted acinar DSL6 tumors express p48 whereas DSL6-derived cell lines, and the tumors induced by them, display a ductal phenotype and lack p48. In human pancreas cancer cell lines and tissues, p48 is present in acinar tumors but not in ductal tumors. Transfection of ductal pancreas cancers with p48 cDNA did not activate the expression of amylase nor a reporter gene under the control of the rat elastase promoter. In some cell lines, p48 was detected in the nucleus whereas in others it was cytoplasmic, as in one human acinar tumor. Together with prior work, our findings indicate that p48 is associated with the acinar phenotype of exocrine pancreas cancers and it is necessary, but not sufficient, for the expression of the acinar phenotype.

The sensitivity of elevated serum ornithine carbamyltransferase (OCT) as an index of hepatotoxicity in rats was assessed in different studies conducted over a number of years and originally designed to examine the toxicity or carcinogenicity of a variety of test chemicals and diets. Changes in serum OCT activities were compared with the more widely used clinical endpoints, alanine aminotranserase (ALT) and aspartate aminotransferase (AST). In the first study, rats received a single oral dose of the hepatotoxic and hepatocarcinogenic fungal toxin aflatoxin B(1) (AFB(1)). The increase in enzyme levels between control and AFB(1)-treated rats was greater for serum OCT than for ALT or AST. This response was similar to the changes in serum enzyme levels in studies where rats ingested a hepatotoxic and hepatocarcinogenic choline deficient (CD) diet. When rats were exposed to the hepatotoxic and nephrotoxic fungal toxin fumonisin B(1) (FB(1)) by intraperitoneal injection for 6 days, serum AST and ALT were significantly elevated above control levels while OCT was unaffected. The peroxisome proliferator ciprofibrate caused elevated ALT and AST but not OCT at week 52 of dietary exposure, after the development of liver nodules and tumours. Of the two liver-specific enzymes examined in all of the studies, ALT was more consistently predictive of hepatotoxicity than OCT.

H-1/N-15 and C-13 NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-N-15]glutamine, [5-N-15]glutamine, [2-N-15]glutamate, (NH4Cl)-N-15, [2-N-15]alanine, and [1-C-13]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. H-1/N-15 NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amidotransfer reaction. In glutamine-free media (NH4+)-N-15 was consumed and incorporated into alanine. (NH4+)-N-15 was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. C-13 NMR revealed that the [1-C-13] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.

Previously performed short-term (4-month) studies demonstrated that vitamins C and E, β-carotene and selenium modulate growth of early putative preneoplastic acinar lesions induced in rat pancreas by azaserine. The present paper summarizes the results of long-term studies performed with azaserine-treated rats maintained on diets high in either β-carotene, vitamins C and E or selenium. It appeared that rats given a diet high in β-carotene, vitamin C or selenium, but not vitamin E, developed fewer pancreatic tumours than controls. The chemopreventive effects of these micronutrients were most pronounced when β-carotene and/or selenium were given during the promotion phase of the carcinogenic process. Surprisingly, cell proliferation in azaserine-induced preneoplastic acinar lesions was higher in rats given β-carotene and/or selenium via the diet in comparison to controls. It is considered unlikely that any antioxidant alone can be associated with protection against cancer. It is concluded that dietary supplementation of combinations of antioxidants may have practical application in chemoprevention of cancer. Copyright (C) 1999 Elsevier Science Ltd.

Although cellular autophagy is recognized as a major pathway of macromolecular catabolism, little data are available regarding its activity or regulation in tumor cells. We approach this problem by morphometrical investigation into the possible changes in autophagic activity during progression of rat pancreatic adenocarcinoma induced by azaserine and promoted by a raw soya flour-containing pancreatotrophic diet. In the present study, the autophagic capacity of the carcinogen-induced premalignant atypical acinar nodule cells was characterized and compared with controls (normal tissue of rats kept on standard laboratory or pancreatotrophic diet and host tissue of the premalignant nodules of the azaserine-treated rats). Given for 90 min, vinblastine, an enhancer of autophagic segregation (i.e. formation of autophagic vacuoles), caused a one to two orders of magnitude larger expansion of the autophagic compartment in atypical nodule cells than in the controls. Then a 20 min blockade of segregation by cycloheximide led to regression of the autophagic compartment, which was barely measurable or moderate in the controls but exceeded 50% in the premalignant cells. At the same time, the cytoplasmic volume fraction of early autophagic vacuoles regressed to a near zero value in each cell type. Expansion and regression rates of these nascent vacuoles showed that both segregation and degradation were 6-20 times faster in the nodule than in normal tissue cells. These results show that the autophagic capacity of the premalignant cells in our system is greatly increased, possibly making these cells unusually sensitive to up-regulation of their self-digesting activity in response to different extracellular signals or drugs.

Carboxymethylating agents are potential sources of endogenous DNA damage that have been proposed as possible contributors to gastrointestinal carcinogenesis. The cytotoxicity of the model DNA carboxymethylating agent azaserine was investigated in human cells. Expression of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) did not affect sensitivity to the drug in two related Raji Burkitt's lymphoma cell lines. DNA mismatch repair-defective variants of Raji cells which display increased tolerance to DNA methylation damage were not selectively resistant to azaserine. Complementary results were obtained with a second carboxymethylating agent, potassium diazoacetate. In contrast, lymphoblastoid cell lines representative of each of the xeroderma pigmentosum complementation groups, including the variant, were all significantly more sensitive to azaserine than nucleotide excision repair-proficient cells. The hypersensitivity of XP cells was not due to systematic differences in the concentrations of intracellular thiol compounds or related thiol metabolizing enzymes. The data indicate that of the two types of potentially lethal DNA damage which azaserine introduces, carboxymethylated bases and O(6)-methylguanine, the former are repaired by nucleotide excision repair and are a more significant contributor to azaserine lethality in human cells.

Previously performed short-term (4-month) studies demonstrated that vitamins C and E, β-carotene and selenium modulate growth of early putative preneoplastic acinar lesions induced in rat pancreas by azaserine. The present paper summarizes the results of long-term studies performed with azaserine-treated rats maintained on diets high in either β-carotene, vitamins C and E or selenium. It appeared that rats given a diet high in β-carotene, vitamin C or selenium, but not vitamin E, developed fewer pancreatic tumours than controls. The chemopreventive effects of these micronutrients were most pronounced when β-carotene and/or selenium were given during the promotion phase of the carcinogenic process. Surprisingly, cell proliferation in azaserine-induced preneoplastic acinar lesions was higher in rats given β-carotene and/or selenium via the diet in comparison to controls. It is considered unlikely that any antioxidant alone can be associated with protection against cancer. It is concluded that dietary supplementation of combinations of antioxidants may have practical application in chemoprevention of cancer. Copyright (C) 1999 Elsevier Science Ltd.

We isolated two new cDNAs, BCH3 and BCH4 (barley C RNA homologues), which are members of the BCH multigene family from barley. BCH genes are thought to encode putative inducible high affinity nitrate transporters. BCH3 was isolated using RACEPCR with specific primers to the BCRNA fragment (Trueman et al., 1996), while BCH4 was isolated using RT-PCR. BCH3 and BCH4 are 1822 and 1705 bp, encode putative polypeptides of 507 amino acids, with a predicted m.w. of 54.6 kDa. Predicted BCH3 and BCH4 proteins are members of a nitrate/nitrite subfamily of major facilitator superfamily. These cDNAs are related to Aspergillus nidulans CRNA, Chlamydomonas rienhardtii NAR3 and NAR4, Hordeum vulgare BCHI and BCH2, and Nicotinia plumbaginifolia NRT2Np. In barley, southern analysis indicates 7–10 copies of BCH genes (Trueman et al., 1996). BCHI, BCH2, BCH3 and BCH4 are highly homologous (greater than 87% identity). In Northern blot analysis, we found that NO 3 − supply to N-deprived plants increased both the abundance of BCH transcripts and 13NO 3 − influx. We analyzed the expression pattern of the four BCH genes using oligonucleotides direct to the 3 ’UTR, and found that all four genes are co-ordinately up-regulated in response to NO 3 − treatment. We assayed the effect of various external concentrations of NO 3 − and found that 50 µM NO 3 − grown plants showed the highest BCH transcript abundance and 13NO 3 − influx. These results provide indirect evidence that the BCH genes encode proteins which are involved in inducible high-affinity transport in plants We analyzed the effect of amino acids feeding on BCH transcript levels, when co-supplied with nitrate, and found that asparagine, aspartate, glutamate and glutamine dramatically decrease transcript levels (>60%). Concurrently, we measured influx using 13NO 3 − and found a different pattern for the amino acid treatments. Glutamine and asparagine decreased influx by 40 and 20% respectively, while aspartate and glutamate decreased influx by 78 and 65%, respectively. The effect of inhibitors of N assimilation were assayed. Tungstate (an inhibitor of nitrate reductase) increased the level of expression of BCH transcripts, whereas there was a slight decrease for methionine sulfoximine (an inhibitor of glutamine synthetase), compared to control (NO 3 − treatment for 6h), but NO 3 − influx decreased by 50%. Azaserine (an inhibitor of glutamate synthase) decreased the levels of BCH transcript by 95% of the control, while NO 3 − influx decreased by >95%. Amino-oxyacetate (an aspartate amino-transferase and glutamate decarboxylase inhibitor) decreased both the BCH transcripts abundance and NO 3 − influx by 65%.

To investigate the effects of increasing concentrations of cholecystokinin octapeptide (CCK-8) on a pancreatic acinar adenocarcinoma.Growth of the tumour was estimated in vivo on rats bearing a subcutaneous pancreatic carcinoma, and in vitro on primary cultured tumour cells. CCK receptors were characterized by binding assays.CCK-8, administered for 12 successive days, exerted a biphasic action on tumour growth: a dose-dependent stimulation with low doses (0.1 and 0.5 microg/kg) and inhibition with high doses (2 and 4 microg/ kg) as shown by respective increases and decreases in tumor volume, protein, RNA and amylase contents. In cell cultures, [3H]thymidine incorporation was dose-dependently increased with 10-(10) to 10(-8) M CCK-8 and inhibited with 10(-7) M. Both effects were completely suppressed by the CCK-receptor antagonists CR 1409 and L 364,718 (10(-4) M). Binding studies showed the overexpression of two classes of CCK-A receptors of low and high affinity when compared to the normal pancreas which was less sensitive to CCK-8.CCK-8 exerts a biphasic growth response on the acinar pancreatic carcinoma, mediated by two classes of CCK-A receptors overexpressed in the tumour.

Activation of protein kinase C (PKC) has been implicated in the high glucose-induced stimulation of matrix protein production in mesangial cells. Since we have found (Kolm-Litty et al., 1998) that glucosamine, similar to the PKC activator phorbol myristate acetate (PMA), mimicks high glucose-induced TGF-beta1 overexpression and subsequent matrix overproduction, the action of these agents on the translocation of PKC isoenzymes was studied in cultured mesangial cells. Exposure to 12 mM glucosamine resulted in rapid and specific translocation of PKC-isoenzymes in mesangial cells i.e. glucosamine caused an increased and sustained translocation of PKC-alpha, -beta and -epsilon while PKC-zeta was essentially unaffected. Comparison with PMA-induced translocation exhibited distinct differences. Exposure to high glucose concentrations of mesangial cells induced translocation of PKC-beta and down-regulation of PKC-epsilon while PKC-alpha and -zeta were essentially unaltered. Presence of azaserine an inhibitor of glutamine: fructose-6-phosphate amidotransferase, the key enzyme of the hexosamine pathway, attenuated the high glucose-induced effects on the membrane fraction of PKC-beta. Our results indicate that i) glucosamine is a potent stimulator of PKC-translocation exhibiting an isoenzyme specific translocation kinetic which is different from PMA-induced PKC-isoenzyme translocation ii) the hexosamine pathway may be possibly involved in the high glucose-induced activation of PKC.

The promoter region of the mouse adenine phosphoribosyltransferase (aprt) gene contains one non-consensus Sp1 binding site at its 5' end followed by three consensus Sp1 binding sites. The two 3'-most binding sites are sufficient for maximal expression of aprt , suggesting that the non-consensus and consensus binding sites at the 5' end are redundant. However, the two 3' sites are not sufficient to block epigenetic inactivation, which led to the hypothesis that the redundant consensus and/or non-consensus 5' Sp1 binding sites are required to block inactivation events. To test this hypothesis, promoter region constructs were made in which the two 5' Sp1 binding sites were mutated alone or in tandem, and then each construct was tested for its ability to withstand epigenetic inactivation. A cis -acting methylation center that is normally located 1.2 kb upstream of the promoter was used to induce inactivation. The results demonstrate that the presence of the redundant consensus Sp1 binding site is required to block methylation-associated gene inactivation. Therefore, the Sp1 binding sites comprising the mouse aprt promoter have evolved two distinct functions, one to promote transcription and the other to block epigenetic inactivation.

Lesch-Nyhan syndrome is a hereditary disorder of purine metabolism causing overproduction of uric acid and neurological problems including spasticity, choreoathetosis, mental retardation, and compulsive self-mutilation. The syndrome is caused by a defect in the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT), which converts guanine and hypoxanthine to the nucleotides GMP and IMP. There is evidence that the neurological problems are due to an adverse effect of the HPRT deficiency on the survival and/or development of dopaminergic neurons, specifically. Here we report that HPRT-deficient PC12 mutants that have a normal or near normal dopamine content (55-97% of that of wild-type cells) fail to undergo neuronal differentiation induced by nerve growth factor (NGF) when the de novo pathway of purine synthesis is partially inhibited. However, nerve growth factor-induced differentiation is near normal under these conditions in PC12 HPRT-deficient mutants containing much lower dopamine levels (

Previous studies revealed that exposure of mesangial cells to high glucose concentration induces the production of matrix proteins mediated by TGF-beta1. We tested if structural analogues of D-glucose may mimic the high glucose effect and found that D-glucosamine was strikingly more potent than D-glucose itself in enhancing the production of TGF-beta protein and subsequent production of the matrix components heparan sulfate proteoglycan and fibronectin in a time- and dose-dependent manner. D-Glucosamine also promoted conversion of latent TGF-beta to the active form. Therefore, we suggested that the hexosamine biosynthetic pathway (the key enzyme of which is glutamine:fructose-6-phosphate amidotransferase [GFAT]) contributes to the high glucose-induced TGF-beta1 production. Inhibition of GFAT by the substrate analogue azaserine or by inhibition of GFAT protein synthesis with antisense oligonucleotide prevented the high glucose-induced increase in cellular glucosamine metabolites and TGF-beta1 expression and bioactivity and subsequent effects on mesangial cell proliferation and matrix production. Overall, our study indicates that the flux of glucose metabolism through the GFAT catalyzed hexosamine biosynthetic pathway is involved in the glucose-induced mesangial production of TGF-beta leading to increased matrix production.

ÖZET Kanser hücrenin nukleusunun hastalığıdır. Çevresindeki normal dokulara göre farklı sitolojik özellikler ve çoğalma davranışları gösteren hücreler kanser hücreleridir. Araştırmamızın amacı, azaserin enjekte edilmiş sıçanların karaciğer ve pankreas hücrelerinde prekanseröz lezyonları ve yağ asitlerinin bu lezyonların oluşumuna etkisi ni ışık mikroskobunda incelemektir. Araştırmamızda outbred dişi Wistar albino sıçanlar, on dört günlük olunca hafta da bir kez intraperitoneal olarak bir doz ( sıçan vücut ağırlığına göre 30 mg/kg ) taze % 0.9 NaCl solüsyonunda eritilmiş azaserin, sonuç konsantrasyonu 3 mg/kg olacak şe kilde enjekte edildi. Bu işlem üç hafta devam etti. Sıçanlar, laktasyon devrinin bitimin de ( yirmi bir gün ) gruplara ayrıldı. Bu gruplar ; I ( n=10 ) Kontrol Grubu 1 ( İşlem görmeyen kontrol sıçanları ), II ( n=10 ) Kontrol Grubu 2 ( % 20 Oleik asit oram % 71.03 olan bitkisel yağ ile beslenmiş grup ), III ( n=10 ) Azaserin enjekte edilmiş grup, IV ( n=10 ) Azaserin enjekte edilmiş ve % 20 Oleik asit oram % 71.03 olan bit kisel yağ ile beslenmiş grup. Sıçanlar on, on sekiz ve yirmi altı haftalık periyodlarda eter ile bayıltıldı. Vücut ağırlıkları kaydedildi. Karaciğer ve pankreas otopsi ile çıkarıldı, ağırlıkları tartıldı. Dokuların farklı bölgelerinden parçalar alındı, fiksatiflere konuldu. Parafine gömülen parçalardan, döner mikrotomu ile 5 um' luk kesitler alındı, hematoksilen-eozin ile bo yandı ve ışık mikroskobunda incelenerek, gereken bölgelerden fotoğraflar çekildi. Elde edilen sonuçlar Kruskal-Wallis One- Way Anova testi ile karşılaştırıldı. İkili kıyaslamalarda Scheffe testi uygulandı. Karaciğer ve pankreas hücrelerinde ; makronukleuslar, çıkıntılı ve değişmiş nukleuslar, hiperkromazi, hücresel ve nuklear pleomorfizm, anormal mitoz bölün meler gibi kanser hücrelerine özgü özellikler bulundu. Ayrıca pankreasda asidofilik ve bazofılik atipikal asinar hücre fokusları görüldü. Sonuç olarak, bulgularımız, azaserin enjeksiyonu ile belirlediğimiz periyod larda pankreas dokusunda incelenen bölgelerde lezyonların başladığını gösterdi. Ayrı ca bu lezyonların karaciğer dokusuna da yayıldığını ortaya çıkardı. SUMMARY Cancer is a disease of the cell nucleus. Cancer cells are show different etiological features and proliferative behaviors from the surrounding normal tissues. The aim of our investigation was to examine the precancerous lesions in the liver and pancreas cells of azaserine-injected rats and the effect of fatty acids in the formation of these lesions by light microscopy. In our study, outbred female Wistary albino rats when fourteen-days-old, were given a single weekly intraperitoneal (30 mg/kg body weight ) dose of azaserine for three weeks, dissolved in freshly 0.9 % NaCl solution to a final concentration of 3 mg/kg on the day of injection. The pups were then returned to their respective dams and allowed to continue suckling until 21 days of age. Rats were divided into groups as below ; I ( n=10 ) Control rats 1 ( untreated ), II ( n=10 ) Control rats 2 ( 20 % w/w oleic acid rates were 71.03 % from vegetable oil ), III( n=10 ) Azaserine- initiated rats, IV ( n=T0 ) Azaserine-initiated rats and 20 % w/w oleic acid rates were 71.03 % from vegetable oil. The rats were fainted with ether at ten, eighteen and twenty-six weekly periods. The weight of body was recorded. The entire rat liver and pancreas was excised at autopsy. The wet weight of each liver and pancreas was recorded. The fragments were cut from different sections in the both tissues and these fragments were put inside the fixatives ( Bouin, Carnoy, 10 % Formalin ). From the fragments embedded in paraffin, slices were cut at 5 um on a Rotary microtome, stained with haematoxylin-eosine and examined by a light microscope. Photographs were taken from the necessary regions. Results were compared with Kruskal-Wallis One- Way Anova and Scheffe test. In the liver and pancreas cells were shown macronuclei, prominent nuclei, bizarre nuclei, hyperkromazi, pleomorphic nuclei and pleomorphic cells, abnormal mitosis, as proper features of cancer cells. In addition, acidophilic and basophilic atypical acinar cell foci were found in the pancreatic tissue. As a result, our findings were shown to started of lesion in the pancreatic cells of azaserine-induced rats at the determined periods. In addition, this lesions were revealed to invasion the liver. 118

Mutants of Escherichia coli and Saccharomyces cerevisiae that lack O6-alkylguanine-DNA alkyltransferase activities have increased spontaneous mutation rates, indicating the presence of a cellular metabolite that can alkylate DNA. Bacterially catalysed nitrosation has been implicated previously in producing the endogenous alkylating agent(s). Here, nitrosated polyamines and azaserine, a model compound for nitrosated peptides, are shown to be mutagenic to E. coli ada ogt mutants deficient in O6-alkylguanine-DNA alkyltransferase activity. The mutagenicity of azaserine may be explained by its ability to methylate DNA, whereas nitrosated spermidine causes DNA damage that is susceptible to both nucleotide excision repair and O6-alkylguanine-DNA alkyltransferase activity, which indicates the generation of more bulky DNA adducts. Nitrosated peptides and polyamines are therefore potential endogenous mutagens that are harmful particularly in O6-alkylguanine-DNA alkyltransferase deficient cells.

The hexosamine biosynthesis pathway and protein kinase C (PKC) activation mediate hyperglycaemia-induced impaired glucose transport, but the relative role of each pathway is unknown. Following a 2 h preincubation of rat adipocytes in the presence of either high glucose (30 mM) plus insulin (0.7 nM) or glucosamine (3 mM), both high glucose and glucosamine inhibited subsequent basal and insulin-stimulated glucose transport, measured at 5.0 mM glucose. Azaserine, an inhibitor of the enzyme glutamine:fructose-6-phosphate aminotransferase, abolished the effect of high glucose, but not that of glucosamine. Ro-31-8220, an inhibitor of PKC, reversed the effects of both high glucose and glucosamine, suggesting that flux through the hexosamine biosynthesis pathway impaired glucose transport acutely by activating PKC. Both high glucose and glucosamine caused a 3-fold increase in PKC activity; this effect of high glucose, but not that of glucosamine, was partially decreased by azaserine. Neither high glucose nor glucosamine altered basal or insulin-stimulated plasma membrane GLUT1 levels, whereas both treatments decreased basal, but not insulin-stimulated, GLUT4 levels. Azaserine abolished the effect of high glucose, but not that of glucosamine, on basal plasma membrane GLUT4 levels. Ro-31-8220, which returned glucose transport to control values, caused a further decrease in plasma membrane GLUT4 levels. It is concluded that, in rat adipocytes, an acute increase in flux through the hexosamine biosynthesis pathway inhibits glucose transport by activation of PKC.

This study was carried out to test the hypothesis that purine nucleotide-generating pathways are required for ligand-stimulated oocyte maturation in meiotically arrested cumulus cell-enclosed oocytes. Oocytes from hormonally primed, immature mice were cultured overnight in Eagle's minimum essential medium containing dibutyryl cyclic AMP (dbcAMP) (to maintain meiotic arrest), plus either mycophenolic acid or alanosine (inhibitors of guanyl and adenyl nucleotide production, respectively). Follicle-stimulating hormone (FSH) was added either at the outset of culture or after a 3-hr preincubation period. Under either of these conditions, the inhibitors suppressed FSH induction of germinal vesicle breakdown (GVB). In addition, the potency of FSH as an inducer of GVB was reduced following the 3-hr preincubation period, but this could be prevented if nucleotide precursors such as hypoxanthine, guanosine, or adenosine were included during the first 3 hr. Furthermore, preincubation had little effect on FSH induction of GVB when hypoxanthine was used to maintain meiotic arrest for the entire culture period. The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, could not mimic this protective effect of hypoxanthine. Azaserine and aminopterin, inhibitors of purine de novo synthesis, blocked hormone-triggered maturation in dbcAMP-arrested oocytes, but had little effect on hypoxanthine-arrested oocytes. The effect of azaserine on dbcAMP-treated oocytes could be reversed by the inclusion of AICA riboside, a compound that can be taken up by cells and phosphorylated to form AICAR, which can enter the purine de novo pathway at a point distal to the sites of azaserine inhibition. FSH was stimulatory to purine de novo synthesis, while azaserine, aminopterin, hypoxanthine, and AICA riboside all suppressed de novo synthesis in the presence or absence of FSH, with dbcAMP having no effect. HPLC analysis of 14C-hypoxanthine metabolism in oocyte-cumulus cell complexes revealed that changes in the pattern of purine metabolism did not mediate the meiosis-inducing effect of FSH. These data support the conclusion that purine nucleotide-generating pathways are vital participants in the mechanism(s) regulating hormone-induced meiotic maturation, and that either the de novo or salvage pathway can fulfill this nucleotide requirement.

The effects of prolonged administration of the diuretic amiloride on pancreatic carcinogenesis induced by azaserine and on the labeling index of carcinogen-induced pancreatic lesions were investigated in Wistar rats. Rats were given 25 weekly injections of 10 mg/kg body weight azaserine and also 5 mg/kg body weight amiloride every other day until the end of the experiment at week 62. Carcinogen-induced pancreatic lesions were examined by histochemical techniques and were classified as ATPase-positive or ATPase-negative. In week 62, quantitative histologic analysis showed that prolonged administration of amiloride significantly reduced the number and size (as percent of parenchyma) of ATPase-positive pancreatic lesions, which are closely correlated with the subsequent development of pancreatic cancer. Amiloride also significantly decreased the labeling index of carcinogen-induced pancreatic lesions, but not of the surrounding acinar cells. In contrast, amiloride has no significant influence on the number and size of ATPase-negative pancreatic lesions. These findings indicate that amiloride inhibits pancreatic carcinogenesis, and that this effect may be related to the reduction of ATPase-positive lesions and to amiloride's inhibition of cell proliferation in neoplastic lesions of the pancreas.

The human leukocyte surface Ag CD38 was recently identified as a nicotinamide adenine dinucleotide (NAD)(+)-glycohydrolase ecto-enzyme, degrading NAD into nicotinamide and ADP-ribose. We show here that expression of CD38 is increased in the Jurkat T cell line after treatment with agents that augment intracellular cAMP, with the permeant cAMP analogue dibutyryl-cAMP (db-cAMP), and also with PMA, which activates protein kinase C. Treatment of human PBL T cells with db-cAMP or submitogenic doses of PMA also increased CD38 expression. Two other nucleotide-hydrolyzing activities were induced on the T cell surface concomitantly with CD38: the human PC-1 molecule, a nucleotide phosphodiesterase/pyrophosphatase that produces AMP from NAD or ADP-ribose, and a nucleotidase that produces adenosine from AMP, but which may be distinct from the CD73 5'-nucleotidase. All three enzymes were up-regulated after stimulation of human peripheral blood T cells with PHA. The coordinated regulation of these ecto-enzymes suggested that, besides a possible signaling function, they may recycle extracellular NAD by degrading it to adenosine and nicotinamide, which can be taken up by cells. In support of this hypothesis, db-cAMP-treated Jurkat cells could degrade extracellular NAD for de novo synthesis of purines, while untreated cells could not. Activated lymphocytes are often located in tissues in which cell death is common. It is suggested that the coordinated expression of these enzymes may allow activated T cells to re-use NAD and nucleotides from dead cells.

In the present study the effects of 0.1 or 1.0 g beta-carotene/kg diet (L beta C or H beta C) and 1.0 mg or 2.5 mg selenium/kg diet (LSel or HSel), as well as combinations of the respective low and high concentrations of beta-carotene and selenium (LMix or HMix) on the initiation/early promotion phase or on the late promotion phase of pancreatic carcinogenesis in azaserine-treated rats, were investigated using cell proliferation and volumetric data of atypical acinar cell foci (AACF) as parameters. The present results indicate chemopreventive effects of dietary selenium, dietary beta-carotene and of their combination on the development of acinar pancreatic lesions induced in rats by azaserine. The inhibitory effect was most pronounced when beta-carotene and/or selenium were added to the diets during the late promotion phase of the carcinogenic process, although inhibition was also observed with these compounds when they were added to the diets during the first 5 weeks of the study only (initiation/early promotion phase). Neither in the initiation/early promotion phase nor in the late promotion phase was a dose-related trend observed. The multiplicities of AACF with a diameter over 1.0 mm and of carcinomas in situ (CIS), as well as the incidence of CIS were not significantly different among the groups. However, in the late promotion experiment a dose-related decline in multiplicity could be observed in the selenium supplemented groups and in the groups receiving combinations of beta-carotene and selenium. Cell proliferation in azaserine-induced AACF, as estimated by the bromodeoxyuridine (BrdU) labeling index, was significantly higher in H beta C, HSel, LMix and HMix groups (initiation/early promotion phase) as well as in H beta C, LSel, HSel, LMix and HMix groups (late promotion phase) than in high fat controls. From the present results it can be concluded that: (i) beta-carotene and selenium have inhibitory effects on pancreatic carcinogenesis induced in rats by azaserine; (ii) the most clear effects were observed when selenium was given as such, or in combination with beta-carotene during the late promotion phase; and (iii) beta-carotene and selenium stimulate cell proliferation in AACF.

Altered cell proliferation is an important characteristic of neoplastic development, hence the quantitation of cell growth in normal and (pre)neoplastic pancreatic tissue will provide useful information on the process (and modulation) of pancreatic tumour promotion in experimental animals. Cell proliferation was estimated by means of BrdU-incorporation in normal pancreatic tissue and in putative preneoplastic pancreatic tissue of azaserine-treated rats and of N-nitrosobis(2-oxopropyl)amine (BOP)-treated hamsters at 2, 6, 12, 26 and 52 weeks post-treatment. Furthermore, the effects of diets high in vegetable oil or fish oil on the BrdU labeling index, the Ki-67 labeling index as well as the apoptotic labeling index were determined in azaserine-induced atypical acinar cell foci (AACF) in the pancreas of rats. In saline- and azaserine-treated rats, the BrdU labeling index (LI) in normal pancreatic acinar cells showed a similar time-related decrease. In azaserine-treated rats, the BrdU-LI in AACF was significantly higher than in normal acinar tissue. The BrdU, Ki-67 and apoptotic labeling indices in pancreatic AACF of rats fed diets high in vegetable oil versus fish oil were 24.8 ± 1.7 vs 15.5 ± 1.2 (BrdU; P < 0.05), 9.9 ± 0.8 vs 6.5 ± 0.5 (Ki-67; P < 0.001) and 1.31 ± 0.12 vs 0.97 ± 0.11 (apoptosis; P < 0.05), respectively. In hamsters, up to 26 weeks after BOP-treatment the BrdU-L1 in acinar cells was significantly higher than in saline-treated animals. In BOP-treated hamsters, ductal cells showed a significantly increased LI 2 weeks post-treatment, whereas the LI in centroacinar cells had significantly increased 2 and 26 weeks post-treatment. In hamster pancreas the LI was significantly higher in tubular ductal complexes than in cystic ductal complexes and was highest in borderline lesions. It is concluded that (i) determination of BrdU-LI provides a reliable parameter to discriminate between putative preneoplastic lesions with a high or low growth potential, hence with a high or low potential to develop into ultimate carcinomas, (ii) BOP causes increased DNA synthesis in normal acinar cells, leading to increased risk of additional DNA damage, implicating the acinar cell to be putatively involved in the development of ductular adenocarcinomas in the hamster pancreas and (iii) quantitation of cell proliferation and apoptosis may be useful to determine the enhancing or inhibitory effects of life-style factors on pancreatic carcinogenesis.

The hormone gastrin is thought to stimulate the growth of certain pancreatic carcinoma cell lines. We have previously detected the presence of the gastrin receptor in rat pancreatic carcinoma cell lines but not in normal rat pancreas. We had not, however, previously demonstrated that gastrin receptor is expressed in pancreatic carcinomas developing in the ratin vivo.Therefore, in the present study, we examined rat pancreatic tissue at various stages in azaserine-induced pancreatic carcinogenesis for gastrin binding and for the presence of gastrin receptor mRNA to determine the temporal expression pattern of the gastrin receptor during thein vivodevelopment of pancreatic cancer. Autoradiography of pancreatic tissue using125I-gastrin-17-I from all azaserine-treated and control animals at 2, 4, 8, and 12 months of age demonstrated no specific gastrin binding. At 18 months of age, normal pancreas, azaserine-induced premalignant pancreatic nodules, and internodular pancreas demonstrated no specific gastrin binding. One of three azaserine-treated animals developed an area of pancreatic acinar cell carcinoma at 18 months of age which exhibited significant specific gastrin binding of 141.8 ± 32.8 fmole/gm of tissue. Southern blot analysis of pancreatic RNA isolated from animals at 12 months of age revealed no gastrin receptor mRNA; however, by 18 months of age, gastrin receptor mRNA was present in all azaserine-treated animals but absent in control animals. In summary, specific gastrin binding is present inin vivoazaserine-induced pancreatic acinar cell carcinoma but absent in normal pancreas and azaserine-induced premalignant pancreatic nodules. Gastrin receptor mRNA is first expressed in azaserine-treated rat pancreas at some point between 12 and 18 months of age. These results demonstrate that expression of gastrin receptor is altered in azaserine-treated rat pancreas and may play a role in the development of pancreatic cancer.

In a previous short-term study (4 months) we found that Sandostatin, when administered prophylactically, inhibited growth of putative pre-neoplastic ductular lesions induced in hamster pancreas by N-nitrosobis(2-oxopropyl)amine (BOP), but not of acinar lesions induced in rat pancreas by azaserine. The present long-term (12 months) study was carried out to investigate the effects of Sandostatin (3 microgram/day), alone and in combination with orchiectomy, on pancreatic carcinogenesis in azaserine-treated rats and BOP-treated hamsters. In order to mimic therapy in humans, treatment of the animals started 4 months after the last injection with carcinogen, when (pre)neoplastic lesions had already developed. After treatment with Sandostatin for 8 months, rats developed fewer pancreatic atypical acinar cell nodules and tumours than those not treated with Sandostatin. Moreover, multiplicity of (pre)neoplastic acinar lesions was also lower in orchiectomized rats than in intact rats. However, Sandostatin treatment did not enhance the inhibitory effect of surgical castration on pancreatic carcinogenesis in rats. In hamsters that were both orchiectomized and treated with Sandostatin, the development of borderline lesions was significantly inhibited, whereas such an effect was not present in hamsters that were either surgically castrated or treated with Sandostatin alone. In Sandostatin-treated hamsters a significantly lower number of microcarcinomas was found than in hamsters not treated with Sandostatin. The present findings suggest that Sandostatin, particularly in combination with surgical castration, might be of therapeutic value for treatment of ductular pancreatic tumours.

In the present study the putative chemopreventive effect of dietary fish oil (MaxEPA) on azaserine-induced pancreatic carcinogenesis in rats was investigated. Groups of rats were maintained on a semipurified low-fat (LF; 5 wt%) diet or on semipurified high-fat (HF; 25 wt%) diets containing 5 wt% linoleic acid (LA) and including 0.0, 1.2, 2.4, 4.7, 7.1 or 9.4 wt% MaxEPA. Animals fed a HF diet developed significantly higher mean numbers of atypical acinar cell nodules (AACNs), adenomas and carcinomas than animals fed a LF diet. Dietary MaxEPA caused a significant (P

In the present study, the expression of the epidermal growth factor receptor (EGFR) was investigated in putative preneoplastic and neoplastic acinar cell lesions induced in the rat pancreas by azaserine, using Northern blotting, in situ hybridisation (ISH) and immunohistochemistry. EGFR protein levels were decreased in putative preneoplastic eosinophilic acinar cell lesions (atypical acinar cell nodules, AACN) in comparison with normal acinar cells of the pancreas. However, EGFR mRNA expression correlated positively with the volume of AACN in pancreatic homogenates and ISH showed equal or stronger EGFR mRNA expression in AACN than in the surrounding normal acinar cells. Neither EGFR protein nor EGFR mRNA was detected in more advanced lesions such as acinar adenocarcinomas (in situ). Moreover, EGFR protein expression showed an inverse relationship with the mitotic rate of the acinar cells. These findings suggest that down-regulation of EGFR at the protein level may abrogate negative constraints on cell growth, which may stimulate the development of putative preneoplastic AACN to more advanced lesions and, ultimately, acinar adenocarcinomas.

In the present study the putative chemopreventive effect of dietary fish oil (MaxEPA) on azaserine-induced pancreatic carcinogenesis in rats was investigated. Groups of rats were maintained on a semipurified low-fat (LF; 5 wt%) diet or on semipurified high-fat (HF; 25 wt%) diets containing 5 wt% linoleic acid (LA) and including 0.0, 1.2, 2.4, 4.7, 7.1 or 9.4 wt% MaxEPA. Animals fed a HF diet developed significantly higher mean numbers of atypical acinar cell nodules (AACNs), adenomas and carcinomas than animals fed a LF diet. Dietary MaxEPA caused a significant (P

In a previous short-term study (4 months) we found that Sandostatin, when administered prophylactically, inhibited growth of putative pre-neoplastic ductular lesions induced in hamster pancreas by N-nitrosobis(2-oxopropyl)amine (BOP), but not of acinar lesions induced in rat pancreas by azaserine. The present long-term (12 months) study was carried out to investigate the effects of Sandostatin (3 μg/day), alone and in combination with orchiectomy, on pancreatic carcinogenesis in azaserine-treated rats and BOP-treated hamsters. In order to mimic therapy in humans, treatment of the animals started 4 months after the last injection with carcinogen, when (pre)neoplastic lesions had already developed. After treatment with Sandostatin for 8 months, rats developed fewer pancreatic atypical acinar cell nodules and tumours than those not treated with Sandostatin. Moreover, multiplicity of (pre)neoplastic acinar lesions was also lower in orchiectomized rats than in intact rats. However, Sandostatin treatment did not enhance the inhibitory effect of surgical castration on pancreatic carcinogenesis in rats. In hamsters that were both orchiectomized and treated with Sandostatin, the development of borderline lesions was significantly inhibited, whereas such an effect was not present in hamsters that were either surgically castrated or treated with Sandostitin alone. In Sandostatin-treated hamsters a significantly lower number of microcarcinomas was found than in hamsters not treated with Sandostatin. The present findings suggest that Sandostatin, particularly in combination with surgical castration, might be of therapeutic value for treatment of ductular pancreatic tumours. Chemicals/CAS: Anticarcinogenic Agents; Azaserine, 115-02-6; Carcinogens; Nitrosamines; nitrosobis(2-oxopropyl)amine, 60599-38-4; Octreotide, 83150-76-9

In the present study the effects of 0.1 or 1.0 g β-carotene/kg diet (LβC or HβC) and 1.0 mg or 2.5 mg selenium/kg diet (LSel or HSel), as well as combinations of the respective low and high concentrations of p-carotene and selenium (LMix or HMix) on the initiation/early promotion phase or on the late promotion phase of pancreatic carcinogenesis in azaserine-treated rats, were investigated using cell proliferation and volumetric data of atypical acinar cell foci (AACF) as parameters. The present results indicate chemopreventive effects of dietary selenium, dietary β-carotene and of their combination on the development of acinar pancreatic lesions induced in rats by azaserine. The inhibitory effect was most pronounced when p-carotene and/or selenium were added to the diets during the late promotion phase of the carcinogenic process, although inhibition was also observed with these compounds when they were added to the diets during the first 5 weeks of the study only (initiation/early promotion phase). Neither in the initiation/early promotion phase nor in the late promotion phase was a dose-related trend observed. The multiplicities of AACF with a diameter over 1.0 mm and of carcinomas in situ (CIS), as well as the incidence of CIS were not significantly different among the groups. However, in the late promotion experiment a dose-related decline in multiplicity could be observed in the selenium supplemented groups and in the groups receiving combinations of β-carotene and selenium. Cell proliferation in azaserine-induced AACF, as estimated by the bromodeoxyuridine (BrdU) labeling index, was significantly higher in HβC, HSel, LMix and HMix groups (initiation/early promotion phase) as well as in HβC, LSel, HSel, LMix and HMix groups (late promotion phase) than in high fat controls. From the present results it can be concluded that: (i) β-carotene and selenium have inhibitory effects on pancreatic carcinogenesis induced in rats by azaserine; (ii) the most clear effects were observed when selenium was given as such, or in combination with β-carotene during the late promotion phase; and (iii) β-carotene and selenium stimulate cell proliferation in AACF.

The biochemical and pharmacological characteristics of specific binding sites for gastrin-releasing peptide (GRP) were investigated in normal exocrine pancreas and in an azaserine-induced pancreatic carcinoma in the rat, under similar experimental conditions. Cells from both types of tissues contained rapid, reversible, temperature-dependent, and highly specific binding sites for GRP. Scatchard analysis of equilibrium data obtained with normal and tumor plasma membranes indicated a single class of high-affinity sites (KD = 0.42 +/- 0.06 and 0.35 +/- 0.05 nM, respectively), but the number of GRP receptors was significantly different (Bmax = 31 +/- 4.5 and 189 +/- 20 fmol/mg protein, respectively). Binding of 125I-GRP1-27 was sensitive to GTP analogues, suggesting that the GRP receptor is functionally linked to a guanyl regulatory protein; however, the wheat germ agglutinin-agarose purified receptor had lost this G-protein activity. Cross-linking of 125I-GRP1-27 either to normal and neoplastic cells or to crude membranes, solubilized membrane proteins, and partially purified receptors revealed the presence of a specific MW 75-kDa polypeptide. N-Glycanase treatment reduced this apparent MW to about 45 kDa. Together, these data suggest that normal and tumor pancreatic cells contain a specific GRP receptor that is expressed more on malignant pancreatic tissues.

In the present study the putative chemopreventive effect of dietary fish oil (MaxEPA) on azaserine-induced pancreatic carcinogenesis in rats was investigated. Groups of rats were maintained on a semipurified low-fat (LF; 5 wt%) diet or on semipurified high-fat (HF; 25 wt%) diets containing 5 wt% linoleic acid (LA) and including 0.0, 1.2, 2.4, 4.7, 7.1 or 9.4 wt% MaxEPA. Animals fed a HF diet developed significantly higher mean numbers of atypical acinar cell nodules (AACNs), adenomas and carcinomas than animals fed a LF diet. Dietary MaxEPA caused a significant (P

In the present study the effects of 0.1 or 1.0 g β-carotene/kg diet (LβC or HβC) and 1.0 mg or 2.5 mg selenium/kg diet (LSel or HSel), as well as combinations of the respective low and high concentrations of p-carotene and selenium (LMix or HMix) on the initiation/early promotion phase or on the late promotion phase of pancreatic carcinogenesis in azaserine-treated rats, were investigated using cell proliferation and volumetric data of atypical acinar cell foci (AACF) as parameters. The present results indicate chemopreventive effects of dietary selenium, dietary β-carotene and of their combination on the development of acinar pancreatic lesions induced in rats by azaserine. The inhibitory effect was most pronounced when p-carotene and/or selenium were added to the diets during the late promotion phase of the carcinogenic process, although inhibition was also observed with these compounds when they were added to the diets during the first 5 weeks of the study only (initiation/early promotion phase). Neither in the initiation/early promotion phase nor in the late promotion phase was a dose-related trend observed. The multiplicities of AACF with a diameter over 1.0 mm and of carcinomas in situ (CIS), as well as the incidence of CIS were not significantly different among the groups. However, in the late promotion experiment a dose-related decline in multiplicity could be observed in the selenium supplemented groups and in the groups receiving combinations of β-carotene and selenium. Cell proliferation in azaserine-induced AACF, as estimated by the bromodeoxyuridine (BrdU) labeling index, was significantly higher in HβC, HSel, LMix and HMix groups (initiation/early promotion phase) as well as in HβC, LSel, HSel, LMix and HMix groups (late promotion phase) than in high fat controls. From the present results it can be concluded that: (i) β-carotene and selenium have inhibitory effects on pancreatic carcinogenesis induced in rats by azaserine; (ii) the most clear effects were observed when selenium was given as such, or in combination with β-carotene during the late promotion phase; and (iii) β-carotene and selenium stimulate cell proliferation in AACF.

Sensitive high performance liquid chromatography techniques, which differentiate between purine and pyrimidine ribonucleoside and deoxyribonucleoside triphosphates, were used to quantify pools in phytohemagglutinin-stimulated T-lymphocytes (98% CD4+ and CD8+) from healthy volunteers. The importance of de novo synthesis and salvage was evaluated by incubating the cells with 14C-radiolabeled precursors (40 microM), azaserine (20 microM; a glutamine antagonist), and ribavirin (50 microM; an IMP dehydrogenase inhibitor). We confirmed that resting T-lymphocytes meet their metabolic requirements by salvage. Noteworthy observations were as follows. First, nucleotide pool expansion over 72 h is disproportionate, with that for purines (ATP and GTP) being 2-fold compared with up to 8-fold for pyridine (NAD) or pyrimidine (UTP, UDP-Glc, and CTP) pools. This supports an additional role for the latter in membrane lipid biosynthesis, protein glycosylation, and strand break repair. Second, intact de novo pathways are essential for such expansion. Azaserine not only inhibited purine synthesis (confirmed by N-formylglycinamide polyphosphate accumulation), but also reduced expansion of pyrimidine and NAD pools by 70%. Ribavirin depleted GTP pools by 40% and reduced pyrimidine pool expansion by 40% at 72 h. These findings underline the importance of pyrimidine ribonucleotide availability as well as GTP synthesis de novo to proliferating T-lymphocytes. They also demonstrate an absence of coordinate regulation between de novo purine and pyrimidine biosynthesis.

A number of integrational vectors were developed for use as genetic tools in the food-borne pathogen Campylobacter coli. Integration of the plasmids occurred following genetic recombination via a Campbell-like mechanism. For an integrative plasmid containing a DNA fragment internal to the C. coli catalase gene, the insertion was mutagenic and led to a catalase-deficient phenotype. A procedure for generating random mutations in the C. coli chromosome, with these suicide-plasmids, was developed. In addition, the construction and utility of an integrable plasmid for generating transcriptional fusions to a cat reporter gene is described.

The mutagenicity of O-diazoacetyl-L-serine (azaserine) and 6-diazo-5-oxo-L-norleucine (DON), glutamine analogues, were assayed in heterozygous soybean plants (Y11y11), according to the appearance of mutational spots (yellow, dark green and twin) on the leaves. The mutagenicity of azaserine was detected at 0.1 mg/ml, and that of DON, at 0.05 mg/ml. DON was strongly cytotoxic at doses exceeding 0.1 mg/ml. After azaserine and DON treatment, large spots which occupied more than half the leaflet were found in the first and second compound leaves. The glutamine analogues increased the frequency of yellow spots much more than dark green spots or twin spots. Light green spots were observed on y11y11 plants. Azaserine and DON induce somatic crossing over, point mutation and segmental loss as major effects.

The present 12-month study was carried out to investigate the effects of the aromatase inhibitor aminoglutethimide, alone and in combination with orchiectomy, on pancreatic carcinogenesis in azaserine-treated rats and N-nitrosobis(2-oxopropyl)amine-treated hamsters. Treatment of the animals started 4 months after the last injection with the carcinogen. They were surgically castrated and/or treated with aminoglutethimide. Aminoglutethimide-treated rats developed less pancreatic tumours than did untreated controls. Multiplicity of (pre-)-neoplastic acinar lesions was lower in orchiectomized rats than in intact rats. Inhibition of pancreatic carcinogenesis was most pronounced in rats that were both orchiectomized and treated with aminoglutethimide. These effects were statistically significant after 8 months, but not after 4 months, of treatment. In hamsters, aminoglutethimide showed an enhancing rather than an inhibitory effect on the formation of ductular pancreatic tumours. Castration appeared to have no effect on the development of N-nitrosobis(2-oxopropyl)amine-induced ductular lesions in the pancreas, either alone, or in combination with aminoglutethimide. The present findings indicate that aminoglutethimide, alone and in combination with surgical castration, might be of value for the treatment of pancreatic acinar tumours, whereas the usefulness of aminoglutethimide for treatment of ductular adenocarcinomas of the pancreas is somewhat doubtful.

The present study was designed to investigate the effects of the carcinogenic agent azaserine on the induction of pancreatic and hepatic polyamine metabolism in rats. One single injection of 30 mg azaserine/kg body weight i.p. is known to induce adenoma and subsequently carcinoma, predominantly in the pancreas, after several months. Male Lewis rats were treated with either azaserine (30 mg/kg body weight i.p.) or saline and 5-10 animals per group were sacrificed 2, 6, 9, 12, 18, 24, and 48 h later. Furthermore, animals were simultaneously treated with the ornithine decarboxylase (ODC) inhibitor alpha-difluoromethylornithine (DFMO) or the polyamine oxidase inhibitor MDL 72527 and killed 6 and 12 h after azaserine injection. The azaserine-induced significant increase in pancreatic putrescine concentrations was accompanied by an increase in spermidine/spermine N1-acetyltransferase but unchanged ODC and was significantly inhibited by N, N'-bis(2,3-butadienyl)putrescine (MDL 72527) but not by DFMO. S-Adenosylmethionine decarboxylase (SAM-DC) activity was significantly decreased in the pancreata of azaserine-treated animals compared to controls. In contrast, the azaserine-induced significant increase in hepatic putrescine was lower and transient, was accompanied by an increase in ODC and SAM-DC, and was completely inhibited by simultaneous DFMO treatment but not by MDL 72527. These data show completely different patterns of activation of polyamine metabolism in the pancreas and in the liver: Azaserine treatment forms putrescine in the liver by de novo synthesis via ODC only, while azaserine-induced pancreatic putrescine is exclusively produced by the interconversion pathway via oxidation of N1-acetylspermidine.

Gut peptides are involved in the growth and carcinogenesis of the exocrine pancreas of rats after treatment with azaserine. However, little is known about the influence of azaserine on expression of gut peptide receptors in the pancreas of the rat. Cholecystokinin, bombesin, somatostatin, secretin and vasoactive intestinal peptide receptors were therefore visualized and quantified by storage phosphor autoradiography in pancreata of either saline control or azaserine-treated rats. As expected, putative preneoplastic lesions were formed in the pancreata of the azaserine-treated but not in the control animals. The pancreata of control rats contained receptors for cholecystokinin, bombesin, somatostatin, secretin and vasoactive intestinal peptide. Cholecystokinin receptors were of the A-type and showed, in contrast to the other receptors, a heterogeneous expression due to variability of the high-affinity receptors. In the pancreata of azaserine-treated animals a significantly increased binding capacity of high-affinity receptors for cholecystokinin was found not only in atypical acinar cell nodules but also in non-nodular pancreas when compared to pancreas of control rats (P < 0.05). Neither atypical acinar cell nodules nor non-nodular pancreas of rats treated by azaserine were shown to possess receptors for the other four types of gut peptide receptors. The spectrum of peptide receptors in pancreas of control and azaserine-treated rats in this study may help to understand the mechanism whereby gut hormones may modulate pancreatic carcinogenesis.

The present 12-month study was carried out to investigate the effects of the aromatase inhibitor aminoglutethimide, alone and in combination with orchiectomy, on pancreatic carcinogenesis in azaserine-treated rats and N-nitrosobis(2-oxopropyl)amine-treated hamsters. Treatment of the animals started 4 months after the last injection with the carcinogen. They were surgically castrated and/or treated with aminoglutethimide. Aminoglutethimide-treated rats developed less pancreatic tumours than did untreated controls. Multiplicity of (pre-)-neoplastic acinar lesions was lower in orchiectomized rats than in intact rats. Inhibition of pancreatic carcinogenesis was most pronounced in rats that were both orchiectomized and treated with aminoglutethimide. These effects were statistically significant after 8 months, but not after 4 months, of treatment. In hamsters, aminoglutethimide showed an enhancing rather than an inhibitory effect on the formation of ductular pancreatic tumours. Castration appeared to have no effect on the development of N-nitrosobis(2-oxopropyl)amine-induced ductular lesions in the pancreas, either alone, or in combination with aminoglutethimide. The present findings indicate that aminoglutethimide, alone and in combination with surgical castration, might be of value for the treatment of pancreatic acinar tumours, whereas the usefulness of aminoglutethimide for treatment of ductular adenocarcinomas of the pancreas is somewhat doubtful.

Gut peptides are involved in the growth and carcinogenesis of the exocrine pancreas of rats after treatment with azaserine. However, little is known about the influence of azaserine on expression of gut peptide receptors in the pancreas of the rat. Cholecystokinin, bombesin, somatostatin, secretin and vasoactive intestinal peptide receptors were therefore visualized and quantified by storage phosphor autoradiography in pancreata of either saline control or azaserine-treated rats. As expected, putative preneoplastic lesions were formed in the pancreata of the azaserine-treated but not in the control animals. The pancreata of control rats contained receptors for cholecystokinin, bombesin, somatostatin, secretin and vasoactive intestinal peptide. Cholecystokinin receptors were of the A-type and showed, in contrast to the other receptors, a heterogeneous expression due to variability of the high-affinity receptors. In the pancreata of azaserine-treated animals a significantly increased binding capacity of high-affinity receptors for cholecystokinin was found not only in atypical acinar cell nodules but also in non-nodular pancreas when compared to pancreas of control rats (P < 0.05). Neither atypical acinar cell nodules nor non-nodular pancreas of rats treated by azaserine were shown to possess receptors for the other four types of gut peptide receptors. The spectrum of peptide receptors in pancreas of control and azaserine-treated rats in this study may help to understand the mechanism whereby gut hormones may modulate pancreatic carcinogenesis.

Background/Aims: Keratin is a member of the intermediate filament family in epithelial cells. Two-dimensional gel electrophoresis of different epithelial cells has shown 20 different keratin polypeptides. Therefore, mapping of the keratin polypeptides can be used to define a specific tissue. Methods: Cytokeratin expression was investigated by using monoclonal antibodies in human surgical specimens and autopsy material of pancreatic, gastric, liver, and colon carcinomas and cholangiocarcinomas, and their metastasis to lymph nodes and liver was examined. In addition, rat acinar cell carcinomas were used to compare cytokeratin expression in ductal vs. acinar cell pancreatic carcinomas. Results: Human pancreatic ductal carcinomas expressed keratins 7, 8, 18, and 19, whereas the majority of rat acinar carcinomas did not express keratins typical for ducts in rat pancreas. The keratin patterns of gastric and colon carcinomas were identical with keratins 8, 18, and 19. In contrast, hepatocellular carcinomas expressed the same keratin pattern as pancreatic acinar carcinomas with keratins 8 and 18, whereas cholangiocarcinomas expressed keratin 7, 8, 18, and 19, similar to pancreatic ductal carcinomas. Metastasis of pancreatic ductal and colon carcinomas retained their keratin patterns. Conclusions: Keratin polypeptide typing of unknown malignant cells can be a useful tool for cell identification.

It has been suggested that linoleic acid (LA) is responsible for the promoting effect of dietary polyunsaturated fat on pancreatic carcinogenesis via an accelerated prostaglandin synthesis, caused by metabolism of LA-derived arachidonic acid in (pre)neoplastic tissue. The purpose of the present study was to investigate whether dietary LA is the cause of pancreatic tumor promotion by a high fat diet. Five groups of 30 azaserine-treated rats and 5 groups of 30 N-nitrosobis(2-oxopropyl)amine-treated hamsters were maintained for 6 months (rats) and 12 months (hamsters) on high fat (25 weight %) AIN diets containing 2, 4, 6, 10, or 15 weight % LA. The results indicated that the strongest enhancing effect on the growth of pancreatic (pre)neoplastic lesions in rats and hamsters was obtained with 4 and 2 weight % of dietary LA, respectively. At higher LA levels the tumor response seemed to decrease rather than increase. In both rats and hamsters the fatty acid profiles of blood plasma and pancreas showed an accurate reflection of the dietary fatty acid profiles: a proportional increase in LA levels was observed in plasma and pancreas with increasing dietary LA. In both species plasma and pancreatic AA levels remained constant, except for arachidonic acid levels in rat plasma, which significantly increased with increasing dietary LA levels. Fatty acid profiles in hamster pancreatic tumors did not differ from fatty acid profiles in nontumorous pancreatic tissue from hamsters fed the same diet. Prostaglandin (PG) E2, 6-keto-PGF1 alpha, PGF2 alpha, and thromboxane B2-concentrations in nontumorous pancreatic tissue were similar among the diet groups. Ductular adenocarcinomas from hamster pancreas showed significantly higher levels of 6-keto-PGF1 alpha, PGF2 alpha, and thromboxane B2, but not of PGE2 in comparison with nontumorous pancreas. It is concluded that the strongest pancreatic tumor promotion by dietary LA is 4 weight % in rats and 2 weight % or less in hamsters, and that PGs may be involved in the development of ductular adenocarcinomas induced in hamster pancreas by N-nitrosobis(2-oxopropyl)amine.

Physical activity (exercise) is a lifestyle factor that has received little attention with regard to its role in the etiology and/or prevention of cancer. These studies examined the effects of treadmill exercise on the early stages of pancreatic carcinogenesis initiated by azaserine in rats. Male Lewis rats were treated with azaserine at 2 weeks of age and weaned to experimental protocols at 3 weeks of age. Two experiments were undertaken; treadmill exercise began at 6 weeks of age (Experiment 1) or at 13 weeks of age (Experiment 2). Rats were exercised for 15-20 min/day and for 3-5 days/week. Treadmill speed and angle of incline were adjusted to afford a range of exercise intensities. The development of putative preneoplastic lesions of the pancreatic acinar cells (henceforth termed foci) was evaluated by quantitative stereological analysis using light microscopy. In Experiment 1, exercise resulted in a known paradoxical reduction in food intake by about 15% of the intake of the sedentary group fed ad libitum. The burden of azaserine-induced foci was decreased by approximately 37%, and this was attributed to the well known effects of reduced caloric intake in these young, rapidly growing rats. In Experiment 2, the higher intensity treadmill exercise group had an increased focal burden, compared to their sedentary pair-fed controls. Importantly, this enhancement occurred despite a reduction in food intake and body fat stores in this treadmill exercise group. These experiments demonstrate that exercise may suppress or promote carcinogenesis, depending upon the stage in the life cycle of the animal.

The pancreatic cancer cell line, Mia PACA, lacks MTAP, and as a result is unable to salvage adenine and methionine, products of methylthioadenosine (MTA) metabolism by MTAP, and is more sensitive to de novo purine synthesis inhibitors (thiopurines, methotrexate, azaserine and L-alanosine). In addition, MTAP deficiency creates a condition where normal, but not tumor tissues, can be protected from 5-fluorouracil (5-FU) toxicity by MTA. In normal tissue, MTA is converted to adenine, consuming 5-phosphoribose-1-pyrophosphate (PRPP) and competitively inhibiting phosphoribosylation of 5-FU (and subsequent cellular retention and inhibition of thymidylate synthase and incorporation into RNA). In MTAP deficient tumors, MTA can not generate adenine; the relative lack of adenine, and abundance of PRPP favors phosphoribosylation of 5-FU to nucleotides. The MTAP gene is linked to the tumor suppressor p16INK4 locus on chromosome 9p21. This locus is frequently deleted in human cancer. As a result, relative or complete MTAP deficiency is common in several human cancers, including pancreatic cancer (33%). We have transfected the Mia PACA cell line with MTAP cDNA and generated three sublines that express MTAP. The parenteral cell line lacking MTAP expression is more sensitive to 5-FU (4 h treatment), and toxicity is not ameliorated by pretreatment with MTA. In contrast, the three sublines expressing MTAP are a log less sensitive to 5FU, when pretreated with MTA. As pancreatic cancer is known to be somewhat responsive to treatment with 5-FU, but doses are limited by toxicity, the protection of the host from toxicity by MTA pretreatment may allow higher doses of 5-FU to be safely given and allow gemcitabine, another antimetabolite used to treat patients with pancreatic cancer, to be added in full doses. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3548. doi:10.1158/1538-7445.AM2011-3548

Although diazo compounds are probably best known for their involvement as versatile intermediates in modern synthetic organic chemistry, a small number of such compounds also occur naturally. Many of the early examples, such as azaserine, originally isolated in the 1950s, have antitumour properties and consist of modified α-amino acids. More recently, other more complex diazo compounds have been isolated from natural sources, and these include diazobenzoquinones, diazonaphthoquinones, such as the SF2415 and A80915 antibiotics, and the diazofluorene-based kinamycins and lomaiviticins. This report will cover the isolation, biosynthesis, biological activity and synthesis of natural products containing a diazo group.

AimGlutamine is the most abundant amino acid in the plasma, and has been shown to exert cardioprotective effects. However, the underlying mechanisms remained unclear, but may include an anaplerotic effect via its metabolic conversion to citric acid cycle (CAC) intermediates or activation of the hexosamine biosynthetic pathway (HBP).MethodsTo assess the potential roles of these mechanisms, we evaluated the metabolic effects of a physiologically relevant concentration of glutamine (0.5mM) in isolated working rat hearts perfused with 13C-labeled substrates with or without 20 M azaserine (an HBP inhibitor) and under restricted supply of carbohydrates (CHO, i.e. without pyruvate/insulin).ResultsWhen perfused with a mixture of CHOs, a fatty acid oleate and insulin (controls), the addition of glutamine had no effect on functional parameters except for a 17% (p0.05) tissue levels of the CAC intermediates (in nmol/gww): citrate: 260 ± 10 vs. 222 ± 6 and malate 128 ± 5 vs. 103 ± 3. Inhibition of HBP with azaserine restored oleate oxidation and tissue CAC levels, but not triglyceride formation. When perfused under restricted supply of CHOs, hearts displayed significantly decreased cardiac output (65%), a greater percent contribution of glucose to pyruvate formation (60%), and lower tissue citrate and malate levels (45%). Addition of glutamine restored cardiac output and glucose contribution to pyruvate formation but not tissue CAC levels.ConclusionCollectively, these results demonstrate the capacity of glutamine to modulate energy substrate selection and function in the perfused heart. Furthermore, the potential mechanisms underlying these effects appear to be mediated via the HBP rather than anaplerosis.

1. 1.Glutamine analogues l -[alphaS,5S]-alpha-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (acivicin) and 6-diazo-5-oxo- l -norleucine (DON) have been shown to possess cytotoxic activity against a wide variety of animal and human xenografted solid tumours, however their potential in man has been limited by toxicity. 2. 2.We have analysed the effects of acivicin and DON on glutamine utilization to determine whether the reason for the disappointing therapeutic profile is solely due to the inefficient inhibition of glutamine metabolism. 3. 3.Human myeloid leukaemic cells treated with acivicin inhibited ribonucleotide biosynthesis but not energy production via glutaminolysis and had little effect on viability, whereas treatment with DON inhibited both ribonucleotide biosynthesis and glutamine oxidation and resulted in reduced viability. 4. 4.Treatment of the myeloid leukaemic cells with the glucose analogue 2-deoxy- d -glucose in addition to DON potentiated the inhibition of de novo nucleotide biosynthesis, glutaminolysis and glycolysis, and caused a further reduction in cell viability. 5. 5.These results provide further support for the essential role of glutamine in cellular metabolism, and indicate that use of the glutamine analogue DON in the treatment of acute myeloid leukaemia may be more clinically effective if used in combination with 2-deoxy- d -glucose.

Using receptor binding assays, we have previously demonstrated the overexpression of the high-affinity cholecystokinin (CCK) receptor and the novel expression of the gastrin (CCK-B) receptor in the azaserine-induced rat pancreatic carcinoma DSL-6. Since cDNA of both the CCK-A receptor (classical pancreatic CCK receptor) coding region and the CCK-B receptor coding region have recently been cloned and sequenced, we investigated the expression of messenger RNA of these receptors in DSL-6 pancreatic carcinoma. Our results showed that the 32P-labelled cDNA probe of the CCK-A receptor coding region hybridized with an approximately 2.7 kb mRNA from both DSL-6 pancreatic carcinoma and normal rat pancreas. However, the relative expression of the CCK-A receptor mRNA in DSL-6 pancreatic carcinoma was approximately 8-fold of that in normal rat pancreas. The 32P-labelled cDNA probe of the CCK-B receptor coding region hybridized with an approximately 2.7 kb mRNA from DSL-6 pancreatic carcinoma; no hybridizing mRNA could be identified from normal rat pancreas. In summary, the CCK-A receptor mRNA is overexpressed approximately 8-fold and the gastrin (CCK-B) receptor mRNA is novelly expressed in DSL-6 pancreatic carcinoma as compared to normal rat pancreas. These results further confirm our previous findings based on receptor binding assays. The gene overexpression of the CCK-A receptor and the novel gene expression of the gastrin (CCK-B) receptor may be generated by alterations in gene regulation during carcinogenesis, and may play an important role in promoting tumor growth.

Cholecystokinin (CCK) has been shown to stimulate the growth of both normal pancreas and azaserine-induced putative preneoplastic pancreatic lesions in the rat. The present study was performed to determine whether these effects are mediated by way of CCK-A receptors, CCK-B receptors, or both. Sixteen-day-old male Lewis rats were given i.p. injections of azaserine at 30 mg/kg body weight. Starting on day 21, rats were given s.c. injections, 5 days/week for 16 consecutive weeks, of either (a) CCK octapeptide (nonselective CCK agonist) (2.50 micrograms/kg body weight, n = 17), (b) tert-butyloxycarbonyl-Trp-Lys(epsilon-N-2-methylphenylaminocarbonyl++ +)-Asp- (N-methyl)-Phe-NH2 (highly selective CCK-A agonist) (1.84 micrograms/kg body weight, n = 18), (c) [(2R,3S)-beta-MePhe28,N-MeNle31]CCK26-33 (highly selective CCK-B agonist) (2.40 micrograms/kg body weight, n = 18), or (d) normal saline solution (control, n = 17). Rats were subsequently sacrificed, pancreatic weights were determined, and quantitative morphometric analysis of atypical acinar cell foci and nodules was performed. Both CCK octapeptide and the selective CCK-A agonist tert-butyloxycarbonyl-Trp-Lys(epsilon-N-2- methylphenylaminocarbonyl)-Asp-(N-methyl)-Phe-NH2 stimulated pancreatic growth and the development of acidophilic atypical acinar cell foci and nodules. Furthermore, the effect produced by the selective CCK-A agonist tert-butyloxycarbonyl-Trp-Lys(epsilon-N-2- methylphenylaminocarbonyl)-Asp-(N-methyl)-Phe-NH2 was greater than that produced by CCK octapeptide. In contrast, the selective CCK-B agonist [(2R,3S)-beta-MePhe28,N-MeNle31]CCK26-33 had no effect. These findings suggest that the growth of putative preneoplastic lesions (acidophilic atypical acinar cell foci and nodules) in the rat pancreas during the early stages of azaserine-induced pancreatic carcinogenesis is mediated specifically by way of CCK-A receptors.