Enzymatic formation of potential anticancer and antiviral inosine analogues.

Theoretically, inosine analogues should act as effective inhibitors of tumor cell proliferation and viral replication. To acquire a broad spectrum of new candidate inosine analogues, a rapid, facile, quantitative and stereoselective method for deaminating potential antitumor and antiviral adenine analogues previously synthesized in our laboratory was developed. A novel 5'-adenylic acid deaminase, with relaxed substrate requirements, from Aspergillus species was utilized to deaminate four hexofuranosyladenine nucleosides and five adenine nucleoside dialdehydes to their corresponding inosine analogues. The fastest rates of deamination for the hexofuranosyl nucleosides were for the compounds where the vicinal hydroxyl groups on the sugars are oriented in the erythro configuration. For rapid deamination of the adenine nucleoside dialdehydes, the R configuration at the proximal carbon atom is preferred, while the nature of the group on the distal carbon atom has no significant effect on the rate or extent of deamination.

Synthesis and antileukemic activity of certain D-fucopyranosyl nucleosides.

Based upon previously discovered antileukemic properties of 9-beta-D-fucopyranosyladenine (1) in cell culture, four new nucleosides containing naturally occurring bases have been prepared from D-fucose. alpha-D-Fucopyranose tetraacetate was condensed with the silylated bases in either acetonitrile or 1,2-dichloroethane with tin(IV) chloride as the catalyst. The intermediate blocked nucleosides were obtained in crystalline form and deacetylated with methanolic sodium methoxide. 1-beta-D-Fucopyranosyluracil (8), 1-beta-D-fucopyranosylthymine (9), 1-beta-D-fucopyranosylcytosine (10) as the hydrochloride salt, and 7-beta-D-fucopyranosylguanine (11) were crystallized, and their structures were verified by spectroscopic techniques. Nucleosides 8 and 9 had only borderline activity against leukemia L1210 cells grown in culture, whereas nucleoside 11 had activity equal to 1. However, nucleoside 10 proved to be twice as active as either 1 or 11. The antileukemic activity, which was due to the inhibition of cell division, was reversible by transfer of the arrested cells to fresh media or by the addition of cytidine.

Angiogenic effects of macrophages isolated from ascitic fluid aspirated from women with advanced ovarian cancer.

The cellular components of ascitic fluid aspirated from the peritoneal cavity of women with advanced ovarian cancer were separated on a Ficoll gradient. Isolated macrophages, which were further purified, elaborated a growth factor which was mitogenic for human endothelial cells isolated from umbilical veins, arteries and the omental microvasculature in vitro, and was angiogenic in vivo. It is postulated that the macrophage-derived factor enhances tumor neovascularization of the widespread ovarian-derived peritoneal malignant lesions appearing in these patients, thus contributing to their rapid growth and metastasis, and the poor prognosis of the disease.

Antiproliferative activity of purine nucleoside dialdehydes against leukemia L1210 in vitro.

Sixteen purine nucleoside dialdehydes were assayed for antiproliferative activity against murine leukemia L1210 grown in vitro. These compounds either lacked the terminal hydroxymethyl group that is necessary in most cases for phosphorylation, and/or had stereochemically different configurations at one or two positions, or had some alteration in the purine ring structure. Among the latter were two lipophilic N6-benzyladenine containing dialdehydes, and two nucleoside dialdehydes with a bromine atom at C-8 of the purine. These nucleoside dialdehydes, unlike most clinically useful anticancer nucleosides, did not require enzymatic phosphorylation to become activated. The most interesting agent in this group of compounds was the lipophilic nucleoside dialdehyde obtained from N6-benzyladenosine after periodate oxidation. It had an IC50 of 1.0 +/- 0.2 microM, and appears to function by limiting the formation of deoxyguanosine diphosphate (dGDP) by inhibition of ribonucleoside diphosphate reductase, the rate limiting step in the biosynthesis of deoxyribonucleotides.

An inhibitor of T lymphocyte blastogenesis derived from the unfertilized ova of Shad (Alosa sapidissima).

Unfertilized ova from Shad, a North Atlantic herring, contains a cytostatic inhibitor of T lymphocyte blastogenesis. The inhibitor has an estimated molecular weight of 10,000-30,000 Da, is heat stable, non dialyzable, and resistant to protease digestion and periodate oxidation. Although the inhibitor functions at an early metabolic event in T lymphocyte mitogenesis, it does not appear to interfere with thymidine transport, antagonize lectin binding to lymphocyte surface receptors, or interfere with the function of an essential serum component in the cell culture media.

Deoxycytidylate aminohydrolase activity in the Novikoff hepatoma is dependent on the localization of the tumor.

The activity of deoxycytidylate aminohydrolase, a pivotal enzyme for pyrimidine biosynthesis in mammalian tissue, is 100x greater in the Novikoff hepatoma harvested from the intraperitoneal cavity than in the same tumor excised from either subcutaneous or intramuscular sites. The increased enzyme activity in the intraperitoneal tumor is not due to an increase in protein synthesis, since there are no significant differences in enzyme activity between normal liver, and either the subcutaneous or intramuscular hepatomas. Evidence is presented which indicates that deoxycytidylate aminohydrolase activity, and the expression of alternate pathways of pyrimidine biosynthesis in the Novikoff hepatoma, is dependent on the localization of the tumor within the host.

Antiproliferative effects of 4',5'-unsaturated adenine nucleosides on leukemia L1210 cells in vitro.

Several 4',5'-unsaturated adenine nucleosides were shown to have antiproliferative activity against L1210 leukemia cells in vitro. The active nucleosides were cytotoxic to the L1210 cells as demonstrated by Trypan Blue uptake. The cytotoxicity was not induced by alterations in the ribonucleoside and deoxyribonucleoside triphosphate levels of the L1210 cells.

A tumor growth inhibitory factor and a tumor growth promoting factor isolated from unfertilized ova of shad (Alosa sapidissima).

In the present study, a cytostatic tumor growth inhibitory peptide and a tumor growth promoting peptide with molecular weights of 20,000-30,000 Da have been identified in the supernatant fraction of unfertilized ova from Shad. The factors can be separated by gel chromatography, thus indicating that the factors are individual molecules. Both of the factors are nondialyzable, heat stable, and resistant to trypsin digestion and periodate oxidation.

Preparation and antileukemic screening of some new 6'-deoxyhexopyranosyladenine nucleosides.

9-beta-D-Fucopyranosyladenine (1) has weak antileukemic activity against L1210 cells grown in culture. Several new 6'-deoxyhexopyranosyladenine nucleosides were synthesized by standard procedures and assayed for activity. The new nucleosides were 9-(6-deoxy-beta-D-glucopyranosyl)adenine (2), 9-(6-deoxy-beta-D-allopyranosyl)adenine (3), 9-(6-deoxy-alpha-L-talopyranosyl)adenine (4), 9-alpha-D-rhamnopyranosyladenine (5), and 9-(6-deoxy-alpha-L-idopyranosyl)adenine (6). In addition, 9-(6-deoxy-alpha-L-sorbofuranosyl)adenine (7) was isolated from the same preparation as 6. None of the new nucleosides 2-7 had activity against L1210 cells in culture. A number of other known nucleosides related in structure to 1 were also tested for activity. One of these, 9-alpha-L-arabinopyranosyladenine, had activity, but was significantly weaker than 1.

Adenosine aminohydrolase activity in the regenerating rat liver.

The specific activity of adenosine aminohydrolase in the regenerating rat liver is significantly increased 12 h after partial hepatectomy. There is a twofold increase in enzyme activity at 48 h, after which the activity begins to decline. However, increased values still persist 7 days postsurgery. The enzyme is located mainly in the soluble supernatant (90-95%) of the cell. The purified enzyme from 48-h regenerating liver and control liver has similar kinetic properties (Km 54-58 microM for adenosine), similar molecular weights (30,000-35,000), and are equally inhibited by an irreversible transition-state analog and a reversible competitive inhibitor. It is concluded that adenosine aminohydrolase in regenerating liver is an integral component of a salvage pathway designed for the reutilization of nucleotides, and thus helps maintain a "growth state" for the regenerating liver.

Trazodone, a nontricyclic antidepressant, is an inhibitor of adenosine deaminase.

Trazodone, a nontricyclic antidepressant drug, is a noncompetitive inhibitor of adenosine deaminase. The specific activity of adenosine deaminase in rat brain cortex homogenate is 140 +/- 15 (SEM) nm/min/gm tissue, while the apparent Km of adenosine is 77 microM. The apparent Ki of trazodone for the brain cortex enzyme is 1 X 10(-4)M, and 6 X 10(-5)M for purified calf intestine mucosal enzyme.

Inhibition of lymphocyte mitogenesis by factor(s) released from macrophages isolated from ascitic fluid of advanced ovarian cancer patients.

Ascitic fluid from women with advanced ovarian carcinomas was shown to contain factor(s) which inhibit(s) T lymphocyte mitogenesis. The factor(s) was (were) demonstrated to be associated with the infiltrating macrophages. The inhibition was reversible and inhibited mitogenesis at some late event in the cell cycle. The inhibitory substance(s) was (were) noncytotoxic, dialyzable, heat-stable at 70 degrees C for 10 min (but unstable at 100 degrees C for 15 min), and partially resistant to protease treatment (55%-70%). Further experiments demonstrated that macrophages isolated from the ascitic fluid of patients with cirrhosis of the liver also released factor(s) which inhibit(s) T lymphocyte mitogenesis. On the basis of our data and data from other investigators, we propose that in advanced human ovarian cancer of epithelial origin, macrophages which infiltrate the ascitic fluid elaborate nonspecific inhibitors of T lymphocyte blastogenesis within the proximal environment, resulting in localized immunosuppression and the subsequent enhancement of metastasis within the peritoneal cavity, the tumor cells themselves being resistant to the cytocidal action of the macrophages due to genetic selection and/or their inherent biochemical ability to circumvent normal immunosurveillance mechanisms. This may account, at least in part, for the rapid metastasis and poor prognosis of human ovarian adenocarcinomas.

Characterization of ellipticine binding to native calf thymus DNA.

The binding of [14C]ellipticine to native calf thymus DNA was studied using equilibrium dialysis. A Scatchard polt revealed the presence of high-and low-affinity binding sites in DNA, the former having a K of 4.0 X 10(7) M(-1) and an n (saturation limiting of binding) of 0.078 (1mol ellipticine/13 mol of DNA nucleotides). The forces involved in stabilizing the high-affinity binding, which has been equated with intercalative binding, were due to a combination of hydrophobic interactions and hydrogen bonding. Difference spectra of ellipticine in the presence of the polydeoxynucleotides, poly d(A-T) or poly d(G-C), showed that there was no base specificity involved in the high-affinity binding. Ellipticine binding to the low-affinity sites, which has been equated with surface binding, was due primarily to the participation of electrostatic interactions of ellipticine with the anionic phosphate groups on the double helical surface of DNA.

Morphological effects of estrogen on the female rat liver nucleolus.

Prolonged administration of nonphysiological amounts of estrogen induced markedly enlarged nucleoli volumes in rat hepatocytes, indicative of increased nucleolar RNA synthesis. Physiological amounts of drug had no apparent morphological effects on the hepatocytes.

Plasma ribonuclease: a marker for the detection of ovarian cancer.

There were no significant differences between the mean blood plasma (leukocyte-free) RNAase activity among 128 healthy women volunteers age 13-70 and 49 women with benign gynecological tumors. Exceptions to this finding were three apparently healthy women volunteers who had plasma enzyme activity which was higher than two standard deviations from the mean of the control subjects. Increased plasma RNAase activity was also demonstrated for 21 of 22 patients with ovarian carcinomas of differential histological types. This group included two patients with Stage IA, two patients with Stage IC ovarian carcinoma, and 17 patients with advanced ovarian carcinoma. The one exception was a patient with a well encapsulated, mucinous cystadenocarcinoma, Stage IA. The plasma RNAase activity returned to normal values in all of the cancer patients who had no clinical evidence of residual malignant tissue after surgical treatment. However, the enzyme activity also returned to a normal value in one of the 17 women in whom all of the malignant tissue was not removed. These data indicate that plasma RNAase activity can be utilized as a tumor marker for the presence of ovarian malignancies of various histological types, and to differentiate between malignant and benign neoplasms.

DNA-dependent protein methylase activity in bull seminal plasma.

The existence of a DNA-dependent protein methylase activity without any concomitant DNA methylase activity was demonstrated in bull seminal plasma. The enzyme utilized S-adenosyl-L-methionine as a methyl donor, and endogenous seminal plasma protein as the substrate. There was no demonstrable enzyme activity when the seminal plasma was preheated at 100 degrees for 10 min, or when the enzyme reaction mixture was incubated at 4 degrees. The protein methylase required a heterologous DNA source, had optimal activity at pH 8.1, and was enhanced in the presence of Mg2+, NH4+, and reduced glutathione. After the methylated protein product was separated from DNA by extraction with 0.2 M HCl, the incorporated radioactivity was shown to be totally solubilized by incubating the protein either with Pronase or 1 M NaOH, while RNase and DNase had no effect. Approximately 70% of the enzymatically synthesized amino acids in the protein product were tentatively identified as O-methylated amino acid ethers by virtue of their elution from a Dowex 50 H+ column with 0.2 M pyridine, and their stability to acid and base hydrolysis. The partially purified methylated product was shown by Sephadex G-50 chromatography to consist of three distinct radioactive proteins with molecular weights of approximately 21,000, 15,000, and 10,000.