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Incubation of cytosolic fractions of some tumour and normal cell lines with 5'-methylthioadenosine (5'-MTA) resulted in the formation of methionine. Methionine formation only occurred in those cell lines possessing 5'-MTA phosphorylase. The kinetics of product formation indicated that 5'-MTA was first rapidly converted into 5-methylthioribose-1-phosphate, followed by its slower conversion into methionine. Methionine synthesis from 5'-MTA was increased in cells previously incubated in methionine-depleted medium supplemented with 0.1 mM L-homocysteine for 24 hr. For most cell lines methionine synthesis from 5'-MTA was linear for only a short time period, and was followed by a first order decrease in the rate of methionine synthesis. Methionine synthesized from 5'-MTA was extensively incorporated into cellular macromolecules suggesting that 5'-MTA may substitute for methionine as a one-carbon source. This was confirmed by growth experiments which showed that low concentrations of 5'-MTA could partially substitute for methionine for some, but not all, cell lines. Higher concentrations of 5'-MTA were growth inhibitory. It may be possible to use 5'-MTA to selectively 'rescue' cells from methionine deprivation produced by the enzyme L-methioninase.

The effect of a number of anti-tumour agents on prostaglandin (PG) production from arachidonate by sheep seminal vesicles has been investigated. Of the drugs examined only those belonging to the alkylating agent type series showed inhibition of enzyme activity. Unlike most inhibitors of PG synthetase (EC 1.14.99.1) these agents caused an inhibition of prostaglandin E2 (PGE2) production, while unaffecting the formation of prostaglandin F 2alpha (PGF2 alpha). This suggests the site of inhibition is the isomerase converting prostaglandin H2 (PGH2) to PGE2. Kinetic studies indicated that merophan [DL-o-micron-(di-2-chloroethylamino)phenylalanine] inhibited the synthetase competitively with respect to substrate. The kinetics of the inhibition were also consistent with the formation of a reversible enzyme-alkylating agent complex prior to irreversible inactivation of the enzyme. The inactivation process could be described by the Main equation from which a dissociation constant (Kd) and a reaction rate constant (k2) were calculated. The inhibition of PG synthetase may be important in the anti-tumour effect of these agents.

The effect of the tumour-bearing state, with or without induced weight loss on host liver fatty acid synthase and acetyl coenzyme A content has been studied in NMRI mice bearing either the cachexia-inducing colon adenocarcinoma (MAC16) or the related tumour (MAC13), which does not produce weight loss. The specific activity of fatty acid synthase was increased in the host liver of animals bearing either tumour and the hepatic content of acetyl CoA was decreased. Animals bearing the MAC16 tumour fed a diet in which 80% of the calories were supplied as medium chain triglycerides (MCT) had depressed fatty acid synthase and increased acetyl CoA levels, similar to those found in non-tumour-bearing controls.

The clinical success of zidovudine has established the human immunodeficiency virus (HIV) reverse transcriptase (RT) as a valid target for the design of drugs to treat acquired immunodeficiency syndrome. In order to facilitate structural studies of this enzyme, expression systems in Escherichia coli, which allow the production of large amounts of RT, have been established. Using this recombinant material the RT has been purified and crystallized. Crystallographic studies currently underway are aimed at elucidating the three-dimensional structure of HIV RT. The availability of a bacterial expression system has enabled structural/functional studies of the RT by site-directed mutagenesis. These studies have identified amino acid residues that are essential for activity of the enzyme and might be involved in substrate binding. It is hoped that structural information of this nature will allow the rational design of HIV RT inhibitors.

Tumors of peripheral tissues contain low levels of succinyl CoA-acetoacetate CoA transferase activity which is not induced in vitro by prolonged cultivation in 2.5 mM DL-3-hydroxybutyrate. Although this enzyme is considered to be the main agent controlling the extent to which ketone bodies serve as metabolic substrates such tumors metabolize D(-)-3-hydroxy[3(14)C]butyrate to 14CO2. Also addition of 3-hydroxybutyrate and/or acetoacetate reduces the amount of 14CO2 produced from D-[U-14C] glucose suggesting a common metabolic intermediate. These observations can be accounted for by the presence of acetoacetyl-CoA synthetase, an enzyme which is able to synthesize acetoacetyl-CoA directly from acetoacetate, ATP and coenzyme A. This is the first demonstration of this enzyme in tumor tissue. The rate of metabolism of acetoacetate by this enzyme is sufficient to account for the production of CO2 from 3-hydroxybutyrate.

The effect of 3-methyl(temozolomide) and 3-ethyl (CCRG 82019) substituted imidazotetrazinones on cytosine methylation has been studied in the human lymphoblastoid cell line GM892. There was a decrease in the 5-methylcytosine content of newly synthesized DNA in cells treated with the 3-methyl and a small increase in cells treated with the 3-ethyl analogue, which was maximal 4 days after drug treatment. There was a progressive decrease in nuclear DNA methyltransferase after treatment with temozolomide with complete inhibition at 11-12 hr after drug addition, followed by a re-establishment of enzyme levels towards control values. While the free drugs had no effect on DNA methyltransferase activity in vitro, DNA isolated from GM892 cells previously treated with temozolomide inhibited the transfer of methyl groups from S-adenosyl-L-methionine to M. lysodeiktious DNA. The maximum effect was observed at 6 hr after drug addition and was proportional to the concentration of temozolomide to which the cells had previously been exposed. These results suggest that temozolomide may induce a block in cellular replication as a result of an indirect inhibition of DNA methylation and cells which escape this block progress with hypomethylated DNA.

Animals transplanted with the MAC16 colon adenocarcinoma showed a loss of body weight as the tumor weight increased, without a reduction in food intake. Both adipose tissue and muscle mass decreased in tumor-bearing animals, although loss of body fat exceeded that of muscle mass for given tumor weight. Urinary nitrogen excretion was significantly elevated when the weight loss did not exceed 3 to 4 g, but above this weight loss there was a conservation of nitrogen and the excretion level fell to or below that found in non-tumor-bearing animals. The presence of a tumor alone was not sufficient to account for the elevated nitrogen excretion, since animals bearing a related colon adenocarcinoma (MAC13) that did not induce weight loss had a nitrogen excretion pattern similar to that of non-tumor-bearing controls. Feeding an isocaloric isonitrogenous diet in which 80% of the calories were supplied as medium chain triglycerides, which significantly elevated plasma levels of ketone bodies, reduced both tumor weight and host weight loss and restored both the nitrogen balance and urea excretion to that of non-tumor-bearing animals. The plasma levels of amino acids, which were reduced in the cachectic state, were also restored to control values in animals fed the medium chain triglyceride diet. These results suggest that excessive nitrogen catabolism in the cachectic state can be prevented by suitable dietary modification.

Human promyelocytic leukaemia HL60 cells were incubated with the glyoxalase intermediate S-D-lactoylglutathione in culture. The effects on cell proliferation, maturation, viability and cell cycle were investigated. When HL60 cells (5 x 10(4)/ml) were incubated with 50-500 microM S-D-lactoylglutathione for two days, the rate of cell proliferation was decreased. This effect was maximal at 500 microM S-D-lactoylglutathione where the cell proliferation rate was only 16% of control levels. There was a concomitant decrease in cell viability but little differentiation. During the first day of treatment, there was a significant decrease in the percentage of cells in the G2-M phase of the cell cycle with a concomitant increase in the G0-G1 phase. In contrast, when HL60 cells were incubated with 1.0-1.5 mM S-D-lactoylglutathione, the inhibition of cell proliferation was progressively lifted, with a concomitant increase in the percentage of differentiated cells (27% differentiation with 1.5 mM S-D-lactoylglutathione). The activities of glyoxalase II and gamma-glutamyl transpeptidase were increased in these cells. S-D-Lactoylglutathione slowly entered the HL60 cells and was consumed over the period when changes in cell cycle distribution, growth arrest and decrease in cell viability were observed. The mechanism of inhibition of proliferation of HL60 promyelocytes by S-D-lactoylglutathione is unknown but it may be related to the ability of S-D-lactoylglutathione to stimulate the assembly of microtubules.

An 'on grid' negative staining method was developed to study the interaction of influenza virus with erythrocyte membranes. The virus was reacted in situ with cells which had been immobilised on an electron microscope grid. Fusion between influenza virus and the membrane of erythrocyte ghosts was clearly seen using this method.

The relative potencies of some standard anti-influenza compounds have been examined in vitro by plaque reduction in calf kidney cells, and in vivo by reduction in virus titers of the lungs of infected mice. Strains belonging to the four subtypes H0N1, H1N1, H2N2, and H3N2 were employed to compare the activity of amantadine hydrochloride and ribavirin. In vitro for all strains except A/NWS/OO(H0N1) amantadine hydrochloride was 3-4 times more active than ribavirin. In vivo with sensitive strains amantadine hydrochloride produced a plateau effect at higher dose levels, but at lower dose levels was marginally more active than ribavirin. The sensitivity of A/Hong Kong/1/68 (H3N2) to amantadine hydrochloride, rimantadine hydrochloride, cyclooctylamine, and ribavirin was also investigated. In vitro rimantadine hydrochloride was similar in activity to amantadine hydrochloride and ribavirin, but cyclooctylamine was considerably less active. In vivo cyclooctylamine was active only at 150 mg/kg/day, whereas rimantadine hydrochloride produced a plateau effect over a range of concentrations from 9,12-150 mg/kg/day.

The extent of macromolecular alkylation by three imidazotetrazinones, 8-carbamoyl-3-(2-chloroethyl)imidazo[5,1-d]-1,2,3,5-tetrazin-4-(3H )-one (mitozolomide) and the 3-methyl CCRG 81045) and 3-ethyl (CCRG 82019) analogues has been studied both in intact cells and with isolated DNA, RNA and protein. Towards isolated DNA and RNA CCRG 81045 was about twice as reactive as mitozolomide and 5-10-fold more reactive than CCRG 82019. Two cell lines were chosen to study macromolecular alkylation, GM892A and Raji, the latter being 10-20-fold less sensitive to mitozolomide and CCRG 81045 than the former, but only one-and-a-half-fold less sensitive to CCRG 82019. Drug uptake into both cell lines was shown to be by a rapid diffusion process with a cell medium distribution ratio not far from unity. For all three agents intracellular radioactivity became associated with macromolecules, and the level found at any time is a balance between the rate of alkylation and the rate of alkyl group removal by repair processes. Both CCRG 81045 and CCRG 82019 produced approximately the same level of alkyl groups bound to DNA, RNA and protein over a 24-hr period, whereas mitozolomide produced a greater extent of alkylation. All three agents left more alkyl groups bound to DNA and RNA in GM892A than in Raji cells, but there was no difference in the level of alkyl groups remaining bound to proteins. However, in GM892A cells the overall level of alkylation of DNA by CCRG 81045 exceeded that of CCRG 82019 only after 24 hr of drug incubation despite the twenty-fold difference in potency of these agents. These results suggest that specific base alkylations rather than total macromolecular alkylation may be more important in determining relative cytotoxicity.

Animals given transplants of the MAC16 colon adenocarcinoma show a progressive decrease in carcass weight as the tumor size increases without a reduction in either fluid or caloric intake when compared with non-tumor-bearing controls. There is a decrease in both carcass fat and muscle mass which is directly proportional to the weight of the tumor. In male animals weight loss occurs when the tumor mass comprises more than 0.3% of the body weight and reaches 30% when the tumor represents 3% of the body weight. There is evidence for the production by the tumor of both lipolytic and proteolytic factors, which may be responsible for the cachexia, since two related mouse adenocarcinomas, which do not produce weight loss, have little lipolytic or proteolytic activity. The lipolytic factor is nondialyzable and is destroyed by both heat and acid. Both insulin and 3-hydroxybutyrate suppress the lipolytic activity of the tumor extract. The MAC16 tumor also contains a serine protease, the activity of which is also completely abolished by insulin and 3-hydroxybutyrate. Animals bearing the MAC16 tumor have an elevated plasma lipolytic and proteolytic activity when compared with non-tumor-bearing controls, suggesting a peripheral effect of the tumor products. The catabolic factors elaborated by the MAC16 adenocarcinoma may be responsible for the loss of both the fat and nonfat carcass mass, but they do respond to normal metabolic controls.

Bacterially expressed recombinant HIV-1 reverse transcriptase is active as both a homodimer of Mr 66,000 subunits and a heterodimer of Mr 66,000 and 51,000 subunits. The heterodimer is formed by cleavage of a C-terminal fragment from one Mr 66,000 polypeptide, which occurs during purification and crystallization of reverse transcriptase. Thus, crystals obtained from purified Mr 66,000 polypeptide preparations consisted of an apparently equimolar mixture of Mr 66,000 and 51,000 polypeptides, which were apparently analogous to the Mr 66,000 and 51,000 polypeptides detected in HIV-infected cells and in virions. Limited proteolysis of the homodimer with alpha-chymotrypsin also resulted in cleavage to a stable Mr 66,000/51,000 mixture, and proteolysis with trypsin resulted in the transient formation of some Mr 51,000 polypeptide. These results are consistent with the reverse transcriptase molecule having a protease-sensitive linker region following a structured domain of Mr 51,000. Further digestion with trypsin resulted in cleavage of the Mr 51,000 polypeptide after residue 223, yielding peptides of apparent Mr 29,000 and 30,000. A minor peptide of Mr 40,000 was also produced by cleavage of the Mr 66,000 polypeptide after residue 223. About half the original Mr 66,000 polypeptides remained resistant to proteolysis and existed in complex with the above peptides in solution. During both chymotrypsin and trypsin digestion there was an increase in the reverse transcriptase activity caused by a doubling of Vmax with little change in Km for dTTP.(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of K562 human erythroleukaemia cells with 8-carbamoyl-3-methylimidazo[5,1-d]-1,2,3,5-tetrazin-4-(3H)-one (CCRG 81045) caused a concentration-dependent increase in the number of cells producing haemoglobin after 3 days of treatment. The ethyl analogue (CCRG 82019) was inactive in the induction of erythroid characteristics. The concentration of 5-methyl-cytosine in the DNA of CCRG 81045 treated cells decreased 3 days after treatment, and was directly proportional to the number of benzidine-positive cells in the cultures, suggesting a direct correlation between hypomethylation of DNA and the induction of haemoglobin synthesis. Although the mechanism of this drug-induced hypomethylation of DNA is not known, the methyl analogue (CCRG 81045) also appeared to reduce template activity of isolated DNA to a greater extent than the ethyl analogue, suggesting that the extent or position of alkylation of DNA bases be important in inhibiting DNA-recognition enzymes.

A comparison has been made of the cachectic effects produced by the transplantable murine adenocarcinoma of the mouse colon (MAC16) with tumour necrosis factor-alpha (cachectin). Tumour necrosis factor-alpha (TNF-alpha) produced a dose-related weight reduction that was accompanied by a decrease in both food and water intake. The degree of weight loss was directly proportional to the decreased food and water intake. In contrast weight loss produced by the MAC16 tumour occurred without a reduction in fluid or nutrient intake. Both the MAC16 tumour and TNF-alpha produced hypoglycaemia and a reduction in the circulatory level of free fatty acids (FFA), but had opposite effects on the level of plasma triglycerides with the MAC16 tumour-induced cachexia causing a decrease and TNF-alpha producing an increase. The MAC16 tumour elaborated a lipolytic factor which caused an immediate release of FFA from adipose tissue. In contrast TNF-alpha had no effect on mobilization of adipose triglycerides over a short time period. Both TNF-alpha and extracts from the MAC16 tumour caused an enhanced release of amino acids from mouse diaphragm, which was suppressible with indomethacin and heat labile. No TNF was detected in the MAC16 tumour or in the serum of tumour-bearing animals. Both tumour and non-tumour-bearing animals responded with a similar elevation of their serum TNF levels 90 min after a single injection of endotoxin. It is concluded that weight loss produced by TNF-alpha arises from an anorexic effect and that this differs from the complex metabolic changes associated with cancer cachexia.