Changes in diacylglycerol and membrane associated protein kinase C activity reflect the growth status of xenografted human mammary carcinoma treated with 8-Cl-cAMP.

The intracellular accumulation of cAMP inhibits the growth of transformed cells in vitro and in vivo, and exposure to various cAMP analogs produces similar results. The influence of such analogs on the growth of neoplastic cells in vivo is less well defined, and the relevance of these analogs for the phosphoinositide pathway has not been established. The present report details the inhibition of tumor growth that occurred when human mammary xenografts were treated with 8-Cl-cAMP, the subsequent rebound in tumor growth that occurred when treatment ceased, and the levels of diacylglycerol and membrane-associated protein kinase C activity that characterized tumors in different growth states. Tumor levels of diacylglycerol and particulate PKC activity appeared to be influenced not only by treatment but also by treatment withdrawal. Changes in these entities tended to coincide with tumor growth rate, being relatively suppressed during growth stasis and markedly elevated during periods of rapid growth. The data presented do not establish a causal relationship. Thus, the concomitant changes noted in tumor growth and tumor levels of either diacylglycerol and membrane-associated protein kinase C may only be coincidental. Alternatively, they may indicate that cAMP analogs inhibit tumor growth in vivo by modulating the phosphoinositide pathway.

Changes in diacylglycerol and cyclic GMP during the differentiation of human myeloid leukemia K562 cells.

When the human myeloid leukemia cell line, K562, was induced to differentiate along the erythroid lineage by a 4 day treatment with 10 microM tiazofurin, the cellular content of diacylglycerol decreased to 35% of the value in untreated control cells. Under the same conditions the content of cGMP decreased to 61% of the control value. Tiazofurin inhibits guanine nucleotide biosynthesis and lowers cellular GTP. When guanosine and adenine were added together with tiazofurin, the differentiation of K562 was prevented, the concentration of diacylglycerol was maintained at control values, and the reduction in the concentration of cGMP was partially prevented. Other inducers of differentiation which acted by different mechanisms, caused similar changes in the concentrations of diacylglycerol and cGMP.

Activation of the respiratory burst in murine phagocytes by certain guanine ribonucleosides modified at the 7 and 8 positions: possible involvement of a pertussis toxin-sensitive G-protein.

The capacity of certain guanine ribonucleosides (modified at the 7 and/or 8 positions) to enhance the respiratory burst of murine peritoneal phagocytes was evaluated. The results show that 8-mercaptoguanosine, 8-bromoguanosine, 7-methyl-8-oxoguanosine and 7-thia-8-oxoguanosine, when injected intraperitoneally into mice, induced peritoneal phagocytes to generate reactive oxygen species as early as 1 h after injection. In vivo administration of the nucleosides induced higher levels of phagocyte activation than in vitro treatment with the same nucleosides. However, the addition of interferon alpha/beta in vitro significantly increased the magnitude of phagocyte activation by the nucleosides, suggesting an important role for cytokines/lymphokines in the nucleoside-induced phagocyte activation in vivo. Furthermore, pre-treatment of phagocytes in vitro with Bordetella pertussis toxin, before treatment with the guanosines, inhibited their capacity to induce the respiratory burst. These observations establish these low-molecular-weight compounds as interesting probes for the study of stimulus-response coupling in phagocytes.

Tiazofurin and selenazofurin induce depression of cGMP and phosphatidylinositol pathway in L1210 leukemia cells.

The synthetic nucleoside tiazofurin(2-beta-ribofuranosylthiazole-4-carboxyamide) and its selenium analog selenazofurin inhibited the growth of L1210 leukemia cell culture in a dose dependent manner with IC50 value of 2.0 and 0.2 Um respectively. The GTP/ATP ratio was diminished 4-6 fold as measured by HPLC, while IMP/ATP increased 6-8 fold. The decreased guanylate pools may explain the 30% reduction in cyclic GMP levels and GTPase activity measured after the treatment with the nucleosides. Inhibition of phospholipase C activity is suggested since diacylglycerol content, protein kinase C activity and phorbol ester binding of the membrane fraction were also reduced 20-40%. These results reveal a novel aspect in the action of these compounds which may play a role in their therapeutic action and selectivity.

Stimulation of phosphoinositide signaling pathway in murine B lymphocytes by a novel guanosine analog, 7-thia-8-oxoguanosine.

A novel guanosine analog, 7-thia-8-oxoguanosine, is shown to induce proliferation in nude mouse splenic lymphocytes in vitro as measured by increased DNA synthesis. Determination of the levels of sn-1,2-diacylglycerol and inositol 1,4,5-trisphosphate in the 7-thia-8-oxoguanosine-treated B lymphocyte cultures revealed an enhanced formation of these second messengers leading to the activation and possible de novo synthesis of protein kinase C. The relevance of these results are discussed in regard to the transduction mechanism linking these guanosine analog generated signals with subsequent B lymphocyte activation events.

Nucleoside and nucleotide modulation of genetic expression--a new approach to chemotherapy.

Unlike conventional enzymes, receptors that activate G proteins do not catalyze the direct formation or cleavage of covalent bonds but act instead as a catalyst for the exchange of GTP vs GDP, which results in major conformational changes in the alpha subunit of G proteins and dissociation and selective binding of the alpha subunit which provokes direct enzyme activation eventually resulting in stimulation of protein kinase A, B or C. Each of these kinases can phosphorylate specific DNA binding proteins which allow new portions of DNA to be read and expressed. Such a series of events can act as switches to control cellular genetic expression resulting in cellular proliferation, differentiation or hormonal secretion of growth factors (Scheme I). Examples of nucleosides and nucleotides which appear to exert their therapeutic effects via G protein control of cellular proliferation resulting in differentiation are tiazofurin, selenazofurin, and 8-chloro-cAMP which have been synthesized and studied in our laboratories. The clinical application of these nucleosides in cancer treatment is presently underway and offers a viable alternative to chemotherapy with highly cytotoxic agents. The use of these derivatives result in down-regulation of the G protein regulatory pathways responsible for rapid cell division. Alternatively, a series of guanosine analogs prepared in our laboratories, 8-bromoguanosine, 8-mercaptoguanosine, 7-methyl-8-oxoguanosine and 7-thia-8-oxoguanosine, all activate various aspects of the immune response by up-regulation of G protein regulatory pathways in various lymphocyte derived cells. Guanosine-like nucleosides which function in this manner could have major clinical application as antitumor, antiviral and antimetastatic agents providing the desired specificity can be achieved. Specific immune enhancement of the aged might be an attainable goal if suitable orally active guanosine derivatives with high specificity can be achieved. The G protein regulatory pathways for modulation of genetic expression in specific cell types provide a major modern approach to new chemotherapeutic agents.

Proteolytic activity in the insulin receptor.

When a highly purified preparation of rat liver insulin receptor is incubated with trypsin, the receptor develops hydrolytic activity towards N alpha-benzoyl-L-arginine ethyl ester, N alpha-p-tosyl-L-arginine methyl ester, and N alpha-benzoyl-DL-arginine-p-nitroanilide, (compounds which are synthetic substrates of trypsin). The activity toward N alpha-benzoyl-DL-arginine-p-nitroanilide is inhibited by soybean trypsin inhibitor but not by N alpha-p-tosyl-L-lysil chloromethyl ketone. These data are consistent with the presence of proteolytic activity in the insulin receptor specific for the bonds whose carbonyl functions are provided by arginine but not by lysine. Furthermore we found that the esterase activity toward N alpha-benzoyl-L-arginine ethyl ester in the presence of trypsin is enhanced by insulin, and that the concentration of insulin that produced the half maximum stimulation is of the same magnitude as the dissociation constant for the soluble receptor. These data suggest that the insulin receptor is a zymogen, activated by trypsin, and based on its specific activity, may be the protease which releases a peptide chemical mediator of intracellular action of insulin.

Synergistic inhibition of growth of breast and colon human cancer cell lines by site-selective cyclic AMP analogues.

Our past studies on the mechanism of cyclic AMP (cAMP)-mediated control of tumor growth, using the experimental rat mammary tumor models as well as human breast cancer cell lines, indicated that the action of cAMP is mediated by the RII cAMP receptor protein, the regulatory subunit of cAMP-dependent protein kinase type II (Y. S. Cho-Chung, J. Cyclic Nucleotide Res., 6: 163, 1980). We now shown that the site-selective cAMP analogues, which are manyfold more active in binding to the cAMP receptor protein than previously studied analogues, demonstrate a potent growth inhibition of seven breast and three colon human cancer cell lines. The cAMP receptor protein has two different cAMP binding sites, and cAMP analogues that selectively bind to either one of the two binding sites are known as either site 1 selective (C-8 analogues) or site 2 selective (C-6 analogues). Nineteen site-selective analogues, C-6 and C-8 monosubstituted and C-6,-8 disubstituted, were tested for their growth regulatory effect. The majority of these analogues demonstrated an appreciable growth inhibition, with no sign of toxicity in all 10 cancer lines at micromolar concentrations. The three most potent inhibitors were 8-Cl-, N6-benzyl-, and N6-phenyl-8-thio-p-chlorophenyl-cAMP, demonstrating 50% growth inhibition at 5-25 microM concentrations (IC50). Furthermore, N6-analogues, in combination with halogen or thio derivatives of C-8 analogues, demonstrated synergistic enhancement of growth inhibition. The growth inhibition paralleled a change in cell morphology, an augmentation of the RII cAMP receptor protein, and a reduction in p21 ras protein. The growth inhibition by 8-Cl-cAMP was not due to its metabolite, 8-Cl-adenosine, since: (a) the growth inhibition by 8-Cl-cAMP was released upon cessation of treatment, whereas that by 8-Cl-adenosine was not released; (b) 8-Cl-cAMP treatment did not affect cell cycle progression, whereas 8-Cl-adenosine brought about G1 synchronization; (c) 8-Cl-cAMP treatment caused reduction of p21 ras protein, whereas 8-Cl-adenosine did not affect p21 levels; and (d) 8-Cl-adenosine was not detected in either cell extracts or medium from the cells treated with 8-Cl-cAMP for 48-72 h. Site-selective cAMP analogues thus provide a new physiological means to control the growth of breast and colon human cancer cells.

Lipid composition of liver plasma membranes from rats intoxicated with carbon tetrachloride.

Rat liver plasma membranes were isolated from rats intoxicated acutely and chronically with carbon tetrachloride and a quantitative analysis of lipids was performed. Membranes from regenerating liver (acute intoxication) are characterized by a 60% drop in total phospholipids with a diminished phosphatidylcholine/phosphatidylethanolamine ratio (PC/PE) and a markedly decreased cholesterol level during the first 3 days after the intoxication, leading to a drastically decreased cholesterol/phospholipid (PL) ratio. In the chronically intoxicated rats (non-regenerating liver), although the phospholipids are diminished, phosphatidylcholine and phosphatidylethanolamine are equally decreased, and therefore the PC/PE ratio is not changed. Cholesterol is not diminished and, since the phospholipids are very low, the ratio cholesterol/PL is increased. These data could be correlated with the membrane fluidity. A decrease in the cholesterol/PL ratio results in a more fluid lipid matrix in the proliferative state of the cell. Treatment with colchicine during chronic intoxication prevented the increase in the cholesterol/PL ratio and improved the clinical conditions of the rats. The modulation of the cholesterol content could be a mechanism to control membrane fluidity during the different physiological states of the cell.

The role of surface charge and hydrophobic interaction in the activation of rat liver adenylate cyclase.

Rat liver plasma membranes were incubated either with procaine, lidocaine or tetracaine to study the binding of glucagon to receptors and the responses of adenylate cyclase to glucagon or fluoride. Procaine treatment increased the glucagon and fluoride activation of the cyclase and the stimulation was concentration-dependent; this compound seemed to act at the G/F unit level since changes in the glucagon binding were not observed and the basal activity was not modified. Tetracaine inhibited the adenylate cyclase activity in the order glucagon greater than basal greater than fluoride; it seems that tetracaine acted at the receptor unit level since it reduced the binding affinity. Tetracaine at high concentration (10 mM) also inhibited the fluoride stimulation of the Lubrol PX-solubilized enzyme; apparently the anesthetic acts on the G/F unit and this would indicate the component is still bound to the catalytic unit. The solubilized enzyme is not longer stimulated by procaine. These data suggested that the F component site of the G/F units is in some aspects different to the G component and more resistant to the detergent. The results of this work allowed a clear distinction among the different components of the glucagon-stimulated adenylate cyclase system and showed the importance of surface charge and hydrophobic interactions as regulatory mechanisms.

Changes in thyroid hormones following liver intoxication with carbon tetrachloride.

The metabolism of thyroid hormones was studied after carbon tetrachloride (CCl4) liver damage. Thyroxine (T4) and triiodothyronine (T3) levels fell and reached a nadir in the first 24 hours after CCl4 administration. Thyroxine binding globulins (TBG) and thyroid stimulating hormone (TSH) also decreased, reaching their lower values in the same period. Blood and liver T4 turnover measured in CCl4 treated rats were faster than in control animals. Therefore, at least three phenomena appear to be involved after CCl4 intoxication in rats: (1) increased utilization and turnover of thyroxine by the regenerating liver; (2) diminished thyroid hormone secretion by the thyroid gland, and (3) reduced concentration of serum iodothyronine carrier proteins. The results support the concept that the liver participates in the metabolic regulation of T3 and T4 which, in turn, control hepatocellular growth.

Colchicine improves the alterations in the liver adenylate cyclase system of cirrhotic rats.

The adenylate cyclase system and the number and affinities of receptors for insulin and glucagon were studied in rats treated with CCl4 and in rats that received colchicine in addition to CCl4. Liver glycogen, cAMP and total collagen content were also measured in those animals. Rats received only mineral oil or only colchicine were used as controls. In this latter group, all parameters measured were normal. Differences in liver collagen content between groups treated with CCl4 and CCl4 + colchicine were not statistically significant. Basal adenylate cyclase activity was 2-fold increased in the CCl4 group. In the animals receiving CCl4 and colchicine, adenylate cyclase activity was normal. Adenylate cyclase activity stimulated by fluoride or by glucagon was also increased in the CCl4 group. However, this increase was due to to the enhanced basal activity. The number of receptors and the affinities of the receptors of glucagon and insulin were normal in all groups. cAMP levels were found increased in the CCl4 treated animals and this was accompanied by a 90% reduction in liver glycogen. In the group treated with CCl4 + colchicine, cAMP was normal and liver glycogen was only reduced 25%. These results suggest that part of the clinical and biochemical improvements observed in the colchicine treated animals were due in part to a reversal of the alterations of the adenylate cyclase system induced by CCl4.

Some enzyme and hormonal attributes of hepatoma cell membranes.

Adenylate cyclase activity was measured in plasma membranes isolated form Morris Hepatomas (44 and 47C) and from their host livers. We found that the enzyme activity in the tumours was very low, approx. 5% of the level in control and host livers. The amount of cAMP and cGMP in the tumours was also lower than in the host livers but the ratio of cGMP to cAMP in the tumours was increased by a factor of 4-5. The membrane binding capacities for the pancreatic hormones insulin and glucagon were measured. Hepatoma membranes bound less glucagon than those of livers. A decrease in the number of the glucagon receptors was found but there were no changes in the affinity constant. For insulin, we found the same binding capacity as the host and control livers; thus there was an increase in the ratio of insulin bound/glucagon bound in tumours as compared to controls. The plasma levels of insulin in the tumour bearing animals were approximately half of those in control, whereas the glucagon levels in plasma were 60-62% higher in tumour bearing animals. These results are discussed in terms of the characterization of normal, foetal and regenerating liver, in comparison with slow growing hepatomas. The levels of cAMP and cGMP are discussed with respect to control mechanisms of cell proliferation.

Hepatocyte growth control: in vitro approach to problems of liver regeneration and function.

Primary monolayer fetal and adult rat hepatocyte culture systems, which are being used to help analyze in vivo mechanisms controlling liver regeneration, proliferation, and differentiation are described. With results from animal studies of normal or genetically altered rats subjected to partial hepatectomy, to chemical infusions, or to specific dietary deficiency regimens, an apparently complex growth regulatory pattern has emerged. The data suggest a working hypothesis postulating interactions among hormone, nutritional, lipoprotein, and novel nucleotide factors at multiple regulatory sites. These findings may provide some conceptual and experimental basis for future research regarding the development of hepatic cancer, as it may arise spontaneously or from exposure to environmental carcinogens.

Development of insulin and glucagon binding and the adenylate cyclase response in liver membranes of the prenatal, postnatal, and adult rat: evidence of glucagon "resistance".

Although plasma glucagon levels in the rat fetus are in the adult range, hepatic glycogen is present in far greater abundance in the fetus than in the adult. To explain this paradox, adenylate cyclase response to glucagon was studied in partially purified membranes of rat livers obtained throughout perinatal life and at 3 months of age. The adenylate cyclase response to glucagon (10(-9) M) was only 7% of the adult response at day 15 of fetal life and 20% on the 21st day. No until after the 30th day postpartum did not reach maturity. Yet, the adenylate cyclase response to stimulation by NaF was comparable to the adult response throughout fetal life. The binding of [125I]iodoglucagon (2 X 10(-9) M) by these membrane preparations was only 1% of the adult level at day 15 of fetal life and increased to 23% at the 21st day, and, like the adenylate cyclase response to glucagon, did not reach maturity until after the 30th day of postnatal life. In contrast, insulin binding on the 15th day of gestation was 11% of the adult level and on the 21st day 45% of the adult level, reaching adult levels by the 30th postnatal day. An increase in membrane-associated particles, reflecting intramembranous protein, was observed during prenatal life, but the mean particle number per mum2 reached adult levels on the 21st day of fetal life, indicating that subsequent changes in hormone binding were clearly independent of non-specific changes in the number of particles. The findings suggest that the fetal liver is less sensitive to glucagon action than the adult liver, and that this glucagon "resistance" is mediated by a reduced capacity of the hepatocyte to bind glucagon at a time when substantial binding of insulin is demonstrable. Selective discrimination against glucagon may be important in promoting the anabolic processes required for normal fetal development.