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To analyze the aberrant expression of cell cycle-related proteins and their biological significance in relation to cirrhosis, we compared the cirrhotic patterns induced by two different types of cirrhotic agents, CCl4 and thioacetamide (TAA) in rats. CCl4 or TAA treatment was given to rats for 8 or 30 weeks, respectively, and the livers were removed at 9, 20, and 30 weeks after the experiment began. The TAA-induced fibrotic pattern was different from the CCl4-induced one, in terms of the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed severe cirrhotic changes at 9 weeks in CCl4-treated rats and at 30 weeks in TAA-treated rats. Immunoblotting for cyclin D1, E, A, B, and proliferating cell nuclear antigen (PCNA) and their counterpart protein kinases (CDK2, 4, and CDC2) showed significant overexpression in rats with severely cirrhotic livers. The p53 tumor suppressor protein increased dramatically in the CCl4-treated group, while it was not detected in the livers of TAA-treated rats. Upregulation of p21WAF1, a CDK inhibitory protein, was detected in TAA-treated rats, but not in CCl4-treated rats. Immunohistochemical data for cyclin D1, E, and PCNA were well correlated with immunoblotting data; these proteins were increased in hepatocytes surrounding the cirrhotic lesions, suggesting that hepatocyte regeneration is correlated with cell cycle-related protein expression in cirrhotic liver. In the TAA-treated rats, the expression of these proteins was increased both in hepatocytes and in ductule cells. Our data suggest that liver cirrhosis induced by CCl4 or TAA is associated with alterations in cell cycle-related proteins, and that the expression of these proteins is responsible for hepatocyte regeneration in the damaged liver and may be involved in liver carcinogenesis. [ABSTRACT FROM AUTHOR]

In order to investigate signal transduction pathways and related changes of actin cytoskeleton organization in cellular senescence, H-ras double mutants--V12S35, V12G37, and V12C40--were constitutively expressed in human foreskin fibroblast (HDF). Senescent HDF cells as well as the H-ras mutant expressers accumulated p-Erk1/2 in the cytoplasm with increased MEK activity and failed to translocate it to nuclei on EGF stimulation. Senescent HDF cells, V12S35 and V12G37 expressers, revealed a failure to export actin fiber from nucleus to cytoplasm and also to form stress fibers. Perinuclear expression of Rac1 was prominent in the HDF cells and V12C40 expresser; however, in the V12S35 expresser, translocation of Rac1 from perinucleus to nucleus and strong expression of RhoA were obvious. In summary, the H-ras double mutant expressers induced premature senescence through the MEK pathway, accompanied by nuclear accumulation of actin and Rac1 proteins, cytoplasmic retention of p-Erk1/2, and marked induction of RhoA expression, suggesting the translocational inefficiency of the intracellular proteins in the senescent HDF cells.

To clarify the sequential changes in pRB and p16 during different stages of hepatocarcinogenesis such as fibrosis, cirrhosis, hepatocellular adenoma (HCA), and hepatocellular carcinoma (HCC), male Fischer 344 rats were singly injected with diethylnitrosamine (DEN), immediately followed with phenobarbital for 1 wk and then thioacetamide (TAA) for 39 wk in drinking water. Rats were killed at 9, 20, 30, and 40 wk after DEN initiation and changes of pRB level, p16 gene hypermethylation, and in vivo gankyrin expression were examined. Histologic examination showed stepwise appearances of fibrosis, cirrhosis, HCA, and HCC at weeks 9, 20, 30, and 40, respectively. Hypermethylation of p16 exon 1 was not found until HCA but appeared in 50% of the rats with HCC accompanied by complete loss of its mRNA expression. The amount of glutathione S-transferase--gankyrin bound to pRB and pRB degradation in the liver depended on the concentration of gankyrin and incubation time. Gankyrin expression preceded pRB degradation in liver cirrhosis. In conclusion, gankyrin expression induced in liver fibrosis accelerated the degradation of pRB during liver cirrhosis, and inactivation of p16 exon 1 by DNA hypermethylation occurred during the progression of tumor cells to poorly differentiated HCC. Inactivation of pRB and/or p16 resulted in complete loss of regulation in the cell-division cycle during early and late stages, respectively, of hepatocarcinogenesis. Mol. Carcinog. 30:138--150, 2001., (Copyright 2001 Wiley-Liss, Inc.)

Background and Aims: Protein kinase C (PKC) plays a key role in the alteration of signal transduction in the liver, which may contribute to the development of liver cirrhosis. The aim of the present study was to examine the subcellular redistribution of PKC isozymes in rat liver cirrhosis, which is induced by two different cirrhotic chemical agents, carbon tetrachloride (CCl4) and thioacetamide (TAA)., Methods and Results: Thioacetamide and CCl4 were administered to rats for 8 and 30 weeks, respectively before rats were killed and autopsies performed at 9, 20 and 30 weeks later. The TAA induced a fibrotic pattern in the liver that differed from that produced by CCl4, notably in the formation of fibrous connective tissue and the proliferation of bile ductule cells. Cholangiofibrosis and clear-cell foci were also observed in TAA-treated rats at 30 weeks. Histological examination revealed that severe cirrhotic changes were present 9 weeks after the commencement of CCl4 treatment and 30 weeks after TAA treatment., Discussion: When the subcellular redistribution of PKC isozymes (PKCalpha, -beta1, -delta, and -epsilon) was examined, all the PKC isozymes in CCl4-treated rats were found to be translocated to the membrane fraction, which may mean PKC activation, and then downregulated by proteolytic degradation after 9 weeks of treatment, which coincided with peak cirrhotic changes. All rats treated with CCl4 recovered to the control level after 20 weeks of treatment. In the case of TAA-treated rats, PKC isozymes were translocated to the particulate fraction of the liver after 9 weeks of treatment and this persisted in most of the rats for the duration of the experiment., Conclusions: From these results, it would appear that PKC translocation preceded morphologic changes, and that an altered subcellular distribution of the PKC isozyme may be associated with the response to liver damage and carcinogenesis.

The effect of nodularin on selective atrophy of left lobes in the liver was investigated in F344 rats. Nodularin was injected for 10 weeks from the third week of initiation with saline or N-nitrosodiethylamine (DEN), grouped as S/N and D/N, respectively. Nodularin significantly decreased weights of left (LL) and caudate (CL) lobes but increased right (RL) and middle (ML) lobes in S/N rats. Activity of protein phosphatases [types 1 (PPI) and 2A (PP2A)] was more severely reduced in S/N than D/N rats; moreover, in LL compared with RL of S/N rats, activity was significantly inhibited by nodularin treatment from week 4, which corresponded to 2 weeks after nodularin injection. However, nodularin significantly induced peroxisomal palmitoyl-CoA oxidase and cytochrome P-450 4A1 expression in S/N compared with D/N rats. An effect of nodularin on apoptosis was evident since expression of Bcl-Xs was clearly induced in LL of S/N rats as opposed to various inductions of Bcl-XL. However, Bcl-XL in RL was persistently induced, with undetectable Bcl-Xs expression. These results demonstrate biochemical evidence of selective atrophy of LL by inhibition of PP1 and PP2A activity, increase of peroxisomal enzymes and induction of Bcl-Xs expression, in contrast to proliferation of RL in rats treated with nodularin alone. However, nodularin endowed DEN-altered hepatocytes with regenerating power and concomitant restoration of phosphatase activity as well as persistent expression of Bcl-XL in D/N rats.

In a previous paper [Lim, Park, Jee, Lee and Paik (1999) J. Cancer Res. Clin. Oncol. 125, 493-499], we showed two major forms of active DNA-6-O-methylguanine:protein-L-cysteine S-methyltransferase (MGMT; EC 2.1.1.63) in the liver with N-nitrosodiethylamine (DEN)-induced carcinogenesis: these were 26 and 24 kDa species. Here we show that a 2 kDa C-terminal fragment was cleaved from the 26 kDa species in vitro by thrombin or microsomal fractions isolated from DEN-treated rat livers. When Ser(204) of the 26 kDa protein was replaced with Ala by site-directed mutagenesis, phosphorylation of the protein was completely abolished, indicating Ser(204) to be the site of phosphorylation. We also show that the phosphorylation was performed by Ca(2+)-independent protein kinase isoenzymes, and that the phosphorylated rat MGMT protein was resistant to digestion by protease(s) whose activity was increased during DEN-induced hepatocarcinogenesis and also by digestion with endopeptidase Glu-C (V8 protease).

The Rbtg3 gene was isolated by PCR (polymerase chain reaction) cloning from the cDNA library of Rat1 fibroblasts that were stimulated with TPA (12-O-tetradecanoylphorbol-13-acetate) or various growth factors for 3h and was found to be a rat homologue of mouse BTG3 and human ANA genes. The Rbtg3 gene had unique DNA sequences in the 5'-UTR and 3'-UTR that contained four ATTTA and one TTATTTA(T/A)(T/A) nonamer motif, and also a polyA addition site. Nucleotide homology of Rbtg3 with BTG3 and ANA was 88.5 and 76.6%, respectively. Expression of Rbtg3 was investigated in SD rats as well as cell lines derived from mouse--SW3T3, NIH3T3 fibroblasts--and rat--Rat1, 3Y1 fibroblasts and PC12--cells. Rbtg3 was highly expressed in brain but barely in lung, kidney, thymus and spleen. The constitutive expression level was high in SW3T3, Rat1 and 3Y1 fibroblasts, but very low in NIH3T3 fibroblast and PC12 cells. However, in all cells tested, Rbtg3 was proved to be one of the primary response genes superinduced by TPA (50ng/ml)+cycloheximide (CHX, 10 microgram/ml). Expression of Rbtg3 was induced by H(2)O(2) (500mM) up to fourfold in PC12 cells and was blocked by pretreatment of NAC (N-acetyl-L-cysteine, 10mM). The induction was ninefold in 3Y1 fibroblasts by menadione (25mM) treatment for 1h, whereas it was reduced to a third of the control level in SW3T3 fibroblast by the same treatment. Rbtg3 was not expressed in NIH3T3 cells but minimally regulated by redox changes as compared with rapid and strong induction of TIS21/BTG2 mRNAs after TPA or H(2)O(2) stimulation. The above results indicate that Rbtg3 is one of many redox-regulated genes as well as a primary response gene.

To investigate how glutathione-S-transferase placental form (GST-P)+ hyperplastic nodules (HNs) are selected and to determine the driving force for progression or regression of HNs, changes in transforming growth factor-beta1 (TGF-beta) and its receptors were examined during hepatocarcinogenesis initiated by N-nitrosodiethylamine (DEN) and promoted by nodularin. The induction of TGF-beta1 expression in the GST-P+ HNs was dependent on nodularin injections for 10 wk, which started the third week after DEN initiation. The kinetics of TGF-beta1 induction during carcinogenesis were quite different from that of simple regeneration after partial hepatectomy (PH): hepatocytes initiated with DEN alone induced TGF-beta1 expression for 24 d, and subsequent stimulation by PH on the fourteenth day after DEN initiation super-induced TGF-beta1 mRNA (50 times that of the control level), as opposed to a transient expression for less than 5 d by PH alone. GST-P+ HNs did not express TGF-beta receptors I (RI) and II (RII) during the early stage of carcinogenesis, whereas the surrounding hepatocytes strongly expressed both of these receptors. On cessation of nodularin injection, however, the expression of RI and RII in the HNs changed significantly: RII+ nodules appeared, and the number and area of RII+/- nodules were significantly increased at 10 wk after the cessation. These findings indicate that induction of TGF-beta expression in GST-P+ HNs might be a strong selection pressure that allows outgrowth of RII- nodules during liver carcinogenesis., (Copyright 1999 Wiley-Liss, Inc.)

Differential expression of DNA-O6MeG: protein-L-cysteine S-methyltransferase (MGMT) activity and posttranslational modification of the protein during liver regeneration and carcinogenesis were compared in Sprague-Dawley male rats after partial hepatectomy and/or single i.p injection of diethylnitrosamine (DEN, 200 mg/kg). Regenerating hepatocytes after partial hepatectomy induced MGMT transiently within 3 days; however, the induction of MGMT was persistent for 2 weeks after DEN injection, and the combined treatment of DEN and partial hepatectomy maintained the elevated MGMT level for up to 4 weeks. The increased activity was transcriptionally regulated, when analyzed by Northern blot hybridization. The major active form of MGMT protein in the partially hepatectomized or DEN-treated rats was a 26-kDa or 24-kDa species respectively, which was confirmed by Western blot analysis and gel slice assay. The biological significance of the differential induction of MGMT during partial hepatectomy or DEN-induced carcinogenesis is not obvious; however, further studies on possible posttranslational modifications of MGMT protein might shed some light on the functional aspect of MGMT induction.

Enzymatic methylation of endogenous proteins in several cancer cell lines was investigated to understand a possible relationship between protein-arginine methylation and cellular proliferation. Cytosolic extracts prepared from several cancer cells (HeLa, HCT-48, A549, and HepG2) and incubated with S-adenosyl-L-[methyl-3H]methionine revealed an intensely [methyl-3H]-labeled 20-kDa polypeptide. On the other hand, cytosolic extracts prepared from normal colon cells did not show any methylation of the 20-kDa protein under identical conditions. To identify nature of the 20-kDa polypeptide, purified histones were methylated with HCT-48 cytosolic extracts and analyzed by SDS-PAGE. However, none of the histones comigrated with the methylated 20-kDa polypeptide, indicating that it is unlikely to be any of the histone subclasses. The [methyl-3H]group in the 20-kDa polypeptide was stable at pH 10-11 (37 degrees C for 30 min) and methylation was not stimulated by GTPgammaS (4 mM), thus the reaction is neither carboxyl methylesterification on isoaspartyl residues, nor on C-terminal farnesylated cysteine. The present study together with the previous identification of N(G)-methylated arginine residues in the HCT-48 cytosol fraction suggests that this novel endogenous 20-kDa arginine-methylation is a cellular proliferation-related posttranslational modification reaction.

We earlier reported that TIS21 mRNA expression was markedly decreased in A549 and NCIH69 human lung cancer cells and in thymic carcinoma tissues obtained from transgenic mice containing simian virus 40 large T antigen (J Cancer Res Clin Oncol 121:279-284, 1995). To determine how TIS21 inhibits growth, we made 293 cells that constitutively expressed TIS21 protein. The constitutive TIS21 expresser lines C9 and C11 grew to a lower saturation density than did those in the vector-transfected clones (V7 and V10) and antisense-transfected clones (AS1 and AS4), and the size of the C9 and C11 cells increased significantly after transfection with TIS21 cDNA. The serum-stimulated cell cycle was analyzed by fluorescence-activated cell sorting after double thymidine treatment; V10 progressed normally through the cell division cycle, but C9 and C11 cells accumulated continuously in G1 phase until 36 h after treatment. On the other hand, the progression of cells that had already entered to S or G2/M phase was not inhibited. When cell-cycle regulatory proteins were measured, C9 and C11 cells showed significantly reduced synthesis of cyclin E and cyclin-dependent kinase (cdk) 4 as well as a decrease in cyclin E-associated cdk activity. These observations led us to conclude that TIS21 overexpression in G1 phase decreased the amounts of cyclin E and cdk4, thereby decreasing the activity of cdks at the G1-S transition.

Recombinant TIS21 protein was overexpressed in Escherichia coli harboring the expression vector plasmid pQE-30 carrying the TIS21 cDNA coding sequence containing an extra 120 nucleotides upstream. Employing this protein consisting of 158 amino acid residues of the main chain plus 40 residues of the fusion peptide. It was found that one of the protein methylase I group [S-adenosylmethionine:nuclear protein/histone-arginine N-methyltransferase; BC 2.1.1.23; J. Biol. Chem., 269, 1075 (1994)] methylated this protein. The methylation products were identified as guanidino-N-methylated arginines. Some of the kinetics of the reaction are described.

Transforming growth factor-beta1 (TGF-beta1) arrests growth and/or stimulates apoptosis of a variety of cells. The biochemical pathways involved in the apoptotic processes, however, remain poorly defined. TGF-beta1 induces DNA fragmentation together with morphological changes, which are characteristic of apoptosis in the FaO rat hepatoma cell line. Histones were remarkably enriched in lysates of these cells during TGF beta1-induced apoptosis. We identified U1-70 kd as a death substrate which is cleaved following TGF-beta1 treatment. The tetrapeptide caspase inhibitor carbobenzoxy-valyl-alanly-aspartyl-(beta-O-methyl)-fluoromethyl ketone (ZVAD-FMK) prevented TGF beta1-induced apoptotic DNA fragmentation and cleavage of the U1-70 kd protein, showing that caspase(s) are involved in TGF beta1-mediated apoptosis. To identify specific caspases involved in apoptosis induced by TGF-beta1 in FaO cells, proteolytic activation of several of these caspases and their substrates were studied as a function of time following TGF beta1-treatment. TGF beta1-treatment induced the progressive proteolytic processing of caspase-2 (ICH-1L/Nedd-2), whereas caspase-1 itself did not show any cleavage from the precursor. Pretreatment with ZVAD-FMK abrogated the maturation of caspase-2 and blocked the apoptotic progress. These results suggest that caspase-2, but not caspase-1, may play a crucial role in TGF beta1-induced apoptosis in these cells.

Administration of TGF-beta1 to both FaO and HepG2 cells significantly induced apoptosis, particularly in FaO cells. Degradation of genomic DNA in FaO cells was rapidly induced by treatment with TGF-beta1 (5 ng/ml) for only 4 hr. 5alpha-dihydrotestosterone (DHT, 25 nM) alone did not affect any significant changes in cell viability and in nuclei of FaO cells; however, pre-treatment with DHT protected genomic DNA degradation induced by TGF-beta1 for 14 hr. Simultaneous treatment with DHT plus TGF-beta1 (D + T) inhibited TGF-beta-induced apoptosis by approximately 50% in FaO cells. On the other hand, D + T treatment increased mitosis in actively growing HepG2 cells. Thus, it is reasonable to conclude that DHT gives growth advantage to hepatocellular-carcinoma cells by inhibiting TGF-beta-induced DNA fragmentation in FaO cells and by inducting mitosis in HepG2 cells.

Protein methylase I (protein-arginine N-methyltransferase) was examined in HCT-48 cells, synchronized by serum deprivation and hydroxyurea treatment. The enzyme activity to methylate the added hnRNP protein A1 increased about 2-fold from G0 to S phase, and then decreased during G2/M phase. The enzymatically [methyl-3H]-labeled hnRNP protein A1 was identified by SDS-PAGE/fluorography, and the products were identified as NG-monomethylarginine and NG,NG-dimethyl-(asymmetric)arginines by HPLC. Among endogenous proteins, the 20-kDa species in the extract was most intensely [methyl-3H]-labeled. This 20-kDa methylation was markedly inhibited by the addition of exogenous hnRNP protein A1, indicating that these two substrates compete for the same protein methylase. The possible role of this post-translational modification has been discussed.

Myelin is a membrane characteristic of the nervous tissue and functions as an insulator to increase the velocity of the stimuli being transmitted between a nerve cell body and its target. Myelin isolated from human and bovine nervous tissue is composed of approximately 80% lipid and 20% protein, and 30% of the protein fraction constitutes myelin basic protein (MBP). MBP has an unusual amino acid at Res-107 as a mixture of NG-monomethylarginine and NG, N'G-dimethylarginine. The formation of these methylarginine derivatives is catalysed by one of the subtypes of protein methylase I, which specifically methylates Res-107 of this protein. Evidence is presented to demonstrate an involvement of this biological methylation in the integrity and maintenance of myelin.

The synaptotagmins together with other vesicle proteins are thought to be essential for the docking and/or fusion of synaptic vesicles with the plasma membrane that occurs following depolarization and calcium influx in presynatic terminals. Syt4, the fourth identified member of the synaptotagmin family, is inducible in PC12 cells by depolarization and secretagogues, and in limbic regions of the adult rat brain by kainic acid-induced seizures. In the present study, we examined the time course of the seizure-induced changes in the expression of Syt4 and Syt1, both in adult animals and during the postnatal period. Syt4 was transiently induced in several structures of the adult rat brain following seizure activity with peak inductions between 4 and 8 h and overal return to control values by 30 h. No induction was observed following seizure activity in 7-day-old animals. The brain regions most sensitive to increased induction were, in decreasing order of sensitivity, hippocampal pyramidal cells dentate granule cells and piriform cortex pyramidal cells. The brain areas showing the greatest Syt4 stimulation in adults were also the areas in which Syt4 was induced by seizures earlier in development. In contrast, Syt1 mRNA was depressed in adult brains following seizure activity, particularly in the dentate granule cells. Our results suggest that the differential regulation of different synaptotagmin genes following excessive neuronal activity might participate in rapid adaptation of subsequent transmitter release.

As a part of a series of investigations on the functions of TIS21 and TIS1 genes, we measured in vivo 12-O-tetradecanoylphorbol-13-acetate (TPA) inducibility of primary response genes (TIS21, TIS8 and TIS1) in the Balb/c mice and the changes of TIS gene expression in thymic carcinoma tissues and A549 and NCIH69 human lung cancer cell lines. In vivo induction of the TIS genes (TIS21, -8 and -1) by intraperitoneal injection of TPA was dramatic only at the needle contact site, i.e. in the abdominal muscle, not in the thigh muscle. Expression of TIS21 and TIS1 in the Balb/c mice thymus, lung, stomach and spleen was very strong (Lim IK et al. 1994a), regardless of TPA injection. Thymic carcinoma tissues developed in SV40-T-antigen-containing transgenic mice did not express TIS21 and TIS1, and expressed TIS8 weakly. Interestingly, induction of TIS21 expression was obliterated in the human lung cancer cells; A549 cells completely lost the ability to express TIS21 after a combined treatment of TPA and cycloheximide. We also measured the induction of TIS genes by TPA and/or cycloheximide in Raw264.7 mouse macrophage cells and U937 human histiocytic lymphoma cells. However, the induction profile was quite different; repressed and deregulated expression in the U937 cells as compared to rapid and transient induction of TIS genes in the Raw264.7 cells. These data may suggest a repressed expression of TIS21 and TIS1 in the cancer tissues and cells derived from the organs that constitutively express TIS21 in mice and in human cancer cells.

Transfection of the Escherichia coli ada gene coding for O6-alkylguanine-DNA alkyltransferase results in expression of ada in mammalian cells and transmission of nitrosourea resistance to cells lacking alkyltransferase activity. We have used a replication-incompetent retrovirus to transfer into mammalian cells a chimeric gene consisting of 548 bp of the promoter-regulatory region of the gene for P-enolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) linked to ada. The PEPCK promoter was used because it is inducible and highly expressed in liver and kidney cells both in vitro and in vivo. After retrovirus infection of the rat kidney cell line, NRK, intact proviral DNA was integrated into the genome of cloned cells. Individual NRK clones produced up to 200 units/mg protein of bacterial alkyltransferase activity compared to 65 units/mg protein of mammalian alkyltransferase in the parent cell line. Transcription of ada mRNA originating from the PEPCK promoter was induced with Bt2cAMP or dexamethasone and the combination caused a 4-fold increase in ada mRNA while total alkyltransferase activity was induced up to 2-fold. NRK clones expressing ada had up to 2.0-fold increased resistance to 1,3-bis(2- chloroethyl)-1- nitrosourea. Thus, retroviral gene transfer of the PEPCKada chimeric gene allows efficient and inducible expression of ada with a resulting increase in alkyltransferase activity and nitrosourea drug resistance. This retrovirus may be used to study the role of alkyltransferase in repair of mutagenic DNA lesions in mammalian cells in vivo.

Transgenic animals expressing genes capable of repairing DNA may be a valuable tool to study the effect of DNA-damaging agents on tissue-specific carcinogenesis. For this reason, we constructed a chimeric gene consisting of the promoter-regulatory region of the phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) gene linked to the Escherichia coli ada gene coding for O6-alkylguanine-DNA alkyltransferase and the polyadenylate region from the bovine growth hormone gene. The PEPCK promoter results in gene expression in liver and kidney and is induced by hormones, and its transcription is regulated by diet. The chimeric PEPCK ada gene was injected into the male pronucleus of fertilized eggs to produce transgenic mice. Six of 65 developing mice contained 5-10 copies of the intact trans gene per genome. Two founders transmitted the trans gene in a heterozygous manner, whereas 3 transmitted as germ line mosaics and 1 did not transmit to F1 offspring. All F1 offspring carrying the PEPCK ada trans gene expressed ada mRNA in liver and kidney and produced a functional alkyltransferase with a protein molecular weight of 39,000 originating from the bacterial gene. Total alkyltransferase activity was increased in the liver of F1 offspring from all founder mice, but offspring of only one founder had elevated renal alkyltransferase levels. A diet high in protein markedly increased ada mRNA and alkyltransferase activity within 1 week in both liver and kidney, whereas a high carbohydrate diet for 1 week markedly reduced expression of PEPCK ada and alkyltransferase levels. Nontransgenic animals were unaffected by these dietary manipulations. During induction with a high protein diet, hepatic alkyltransferase in transgenic mice was 16.6 +/- 1.5 units/micrograms DNA (mean +/- SE) compared to 5.3 +/- 0.6 units/micrograms DNA in control animals. This level of alkyltransferase is higher than that in any mammalian tissue noted previously except human liver. Transgenic animals expressing high levels of alkyltransferase should help define the role of DNA repair in protection from carcinogenesis induced by N-nitroso compounds.

A non-washout preparation using Picolax in 40 patients, compared with a standard method using washouts in 38 patients, showed no significant difference in the contamination by residual formed or semiliquid faeces, although Picolax was associated with significantly more contamination due to adherent faeces and mucus. The overall differences in contamination were most marked in the transverse colon. Picolax also affected the smooth muscle tone of the bowel wall, causing an increase in 'mucosal crinkling'.

Maloney murine leukemia virus-based, replication-defective retroviral vectors containing the neomycin resistance gene (neo) were developed to transfer the Escherichia coli ada gene coding for O6-alkylguanine-DNA alkyltransferase, into mammalian cells. To optimize gene transfer and expression, the following promoters were linked to ada: the Maloney murine leukemia virus promoter within the long-terminal repeat, the Rous sarcoma virus promoter, the thymidine kinase promoter, or the human phosphoglycerate kinase promoter. Sequences were transfected into the helper virus-free retroviral packaging psi-2 cell line. Recombinant retroviruses were tested in CCL-1 cells, which, like most murine tissues, have low levels of alkyltransferase and are sensitive to 1,3-bis(2-chloroethyl)nitrosourea (BCNU), and in NIH-3T3 cells, which are BCNU resistant and have high levels of alkyltransferase. Lines infected with each of the four retroviruses were selected for neo expression and found to have intact proviral integration and ada gene expression. Alkyltransferase activity was greatest with retrovirus containing the Rous sarcoma virus-ada gene; infected NIH-3T3 cells had up to 2300 units of alkyltransferase/mg of protein compared with 151 units/mg of protein in control cells, and infected CCL-1 cells had up to 1231 units/mg of protein compared with 33 units/mg of protein in control cells. CCL-1 cells expressing ada were more resistant to BCNU cytotoxicity than were controls. However, NIH-3T3 cells expressing ada were only slightly more resistant to BCNU than controls, possibly because most of the ada protein was cytoplasmic rather than nuclear as suggested by immunohistochemical stain. These studies establish a series of retroviruses containing the bacterial ada gene, which efficiently infect mammalian cells. ada expression increases nitrosourea resistance in cells with low mammalian alkyltransferase activity.