Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors.

Septic shock results from excessive stimulation of the host immune system, especially macrophages, by lipopolysaccharide (LPS), or endotoxin, which resides on the outer membrane of bacteria. Protein tyrosine kinase inhibitors of the tyrphostin AG 126 family protect mice against LPS-induced lethal toxicity. The protection correlates with the ability of these agents to block LPS-induced production of tumor necrosis factor alpha (TNF-alpha) and nitric oxide in macrophages as well as LPS-induced production of TNF-alpha in vivo. Furthermore, this inhibitory effect correlated with the potency of AG 126 to block LPS-induced tyrosine phosphorylation of a p42MAPK protein substrate in the murine macrophage.

Inhibition of phorbol ester-mediated interleukin-2 production by cellular differentiating agents.

Lymphocyte proliferation induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) is inhibited by agents known to induce differentiation in murine erythroleukemia cells and other cell lines. In the present study, we determined the cellular targets for the action of TPA among murine thymocyte subpopulations, the phase of blastogenesis that is activated by the tumor promoter, and the phase that is inhibited by the differentiating agents. Mouse thymocytes were fractionated into populations bearing receptors for peanut agglutinin (PNA; PNA-positive cells) and populations lacking such receptors (PNA-negative cells). TPA is comitogenic for lectin-treated, unfractionated thymocytes and PNA-negative thymocytes but not for PNA-positive thymocytes. PNA-negative cells, a minor population in unfractionated thymocytes, are therefore the cellular targets for the comitogenic activity of TPA. TPA induces the production of interleukin-2 (IL-2) in lectin-treated PNA-negative populations but not in PNA-positive cells. The differentiating agents inhibit TPA-mediated proliferation of unfractionated and PNA-negative, lectin-treated thymocytes. In contrast, IL-2-mediated proliferation of lectin-treated thymocyte subpopulations is resistant to inhibition by these agents. Inhibition appears to be related to decreased production of IL-2, since the differentiating agents inhibit IL-2 production by both PNA-negative thymocytes and by a human leukemic cell line.

Tyrphostin 4-nitrobenzylidene malononitrile reduces chemotherapy toxicity without impairing efficacy.

In mice, 4-nitrobenzylidene malononitrile (AG1714), which belongs to the tyrphostin family, reduced toxicity induced by doxorubicin and cisplatin without impairing their antitumor efficacy. AG1714 reduced mortality induced by doxorubicin and cisplatin. It prevented, in a dose-dependent manner, cisplatin-induced nephrotoxicity as assessed by measurement of serum creatinine and blood urea nitrogen levels. The protective effect of AG1714 was most pronounced on its administration 2 h before cisplatin. AG1714 also prevented doxorubicin-induced myelosuppression as assessed by the scoring of bone marrow nucleated cells and colony-forming units. Cisplatin-induced small intestinal injury was also protected by AG1714 as assessed by histopathological analysis. In vitro, AG1714 reduced cisplatin-induced apoptosis in a murine fibroblastic cell line (A9) and did not affect doxorubicin-induced apoptosis of B-16 melanoma cells. In contrast to its protective effect against mortality and injury of normal tissues induced by chemotherapy, AG1714 did not impair its antitumor activity and in some tumor models enhanced it. This was evident by using the murine tumors B-16 melanoma, Lewis lung carcinoma, and methylcholanthrene-induced fibrosarcoma and the human tumors SK-28 melanoma and human ovary carcinoma xenografts in nude mice. Experiments in which low and high doses of cisplatin and doxorubicin were administered to tumor-bearing mice demonstrated that AG1714 reduced mortality of high-dose chemotherapy and increased its therapeutic index. AG1714 could provide a novel, useful tool to improve chemotherapy by allowing dose intensification.

Antibodies to neuroblastoma cell line proteins in patients with schizophrenia.

The presence of antibodies against neural antigens was investigated in the serum of patients with schizophrenia, major depression and normal controls. Different immunological abnormalities, humoral and cellular, were reported in schizophrenia and major depression. The pathogenesis of schizophrenia is multifactorial. An autoimmune mechanism was suggested as a possible factor. We tested the serum of 26 patients with schizophrenia, eight patients with major depression and 22 normal controls. The serum samples were tested for antibody binding to protein extracts of IMR-32 neuroblastma cell line using Western blot analysis. Immunoglobulins of eight patients with schizophrenia (30.71%) reacted with a protein of 80-85 kDa. Serum samples from subjects of other groups did not react with this protein. Sera of all patients with major depression but one, and all normal controls reacted with HSP 60 kDa to different extent. This is an apparent discrepancy with the findings of Kilidireas et al. [Kilidireas, K., Latov, N., Strauss, D.H., Gorig, A.D., Hashim, G.A., Gorman, J.M., Sadig, S.A., 1992. Antibodies to the human 60 kDa heat shock protein in patients with schizophrenia. Lancet 340, 569-572.] who demonstrated the presence of antibodies against HSP 60 kDa in 44% of patients with schizophrenia tested and 8% of normal subjects. HSP 60 kDa is an antigen of many pathogens and antibodies against it might be a result of an infection and cannot be a good indicator for an autoimmune process. The presence of antibodies against a protein of 80-85 kDa should be investigated as a possible specific indicator.

Tyrphostins reduce chemotherapy-induced intestinal injury in mice: assessment by a biochemical assay.

Intestinal injury that results from chemotherapy belongs to the major factors of dose-limitation in tumour therapy. The tyrphostins AG1714 and AG1801 reduce cisplatin and 5-FU-induced small intestinal mucosal damage, using a quantitative biochemical assay. The assay is based on the determination of the enzymatic activity of gamma-glutamyl transpeptidase, a marker of the brush border epithelium of the small intestine.

Hemin-induced cap formation in lymphocytes: inhibition by protein tyrosine kinase inhibitors.

Hemin, an oxidant which is mitogenic for lymphocytes, was found to induce cap formation for Con A sites in murine and human lymphocytes and for IgG and Thy 1.2 sites in murine lymphocytes. Doses of hemin which induced capping also induced a redistribution of actin to a detergent-insoluble form. Similar to hemin, we found that heat shock also induced capping of Con A and IgG sites in murine splenocytes, as well as actin redistribution in human peripheral blood mononuclear cells. Specific inhibitors of protein tyrosine kinases, termed tyrphostins, were found to inhibit hemin-induced cap formation. In addition, ligand-dependent cap formation was also inhibited by tyrphostins. Hemin-induced cap formation may result from alteration in cytoskeletal structure and distribution. In addition, changes in membrane lipid composition induced by phospholipase C-gamma 1, known to be activated by protein tyrosine phosphorylation, may initiate actin polymerization and membrane-site redistribution.

Osmotic enhancement of mitogenic stimulation in PNA-negative murine thymocytes.

Increasing the osmolarity of the culture medium enhances the response of peanut agglutinin (PNA)-negative thymocytes to stimulation by 12-O-tetradecanoylphorbol-13-acetate (TPA), interleukin 1 (IL-1) and interleukin 2 (IL-2) in the presence of phytohemagglutinin (PHA). The effect was attained by the addition to the medium of salts such as NaCl and KCl or by addition of nonionized compounds such as sucrose and fucose. The enhanced response was monitored by determination of [3H]thymidine incorporation, IL-2 production, and blasts formation. The potentiating effect of hypertonic medium on PNA-negative thymocytes treated with PHA and TPA was most pronounced at suboptimal concentrations of PHA. Hypertonic medium did not enhance the response of thymocytes treated with TPA and supraoptimal concentrations of PHA. Increasing the osmolarity of the medium 44 hr after initiation of culture did not enhance [3H]thymidine incorporation in thymocytes that were pulsed between 52 and 72 hr. The enhancing effect of increased osmolarity in mitogenic stimulation of thymocytes may be related to osmotic activation of the Na+/H+ antiport.

De novo synthesis of purine nucleotides in human blood platelets.

Human blood platelets were found to carry the complete pathway of de novo purine nucleotide synthesis. The rate of purine synthesis was gauged by the rate of incorporation of precursor (14C)formate into purines. The effect on formate incorporation of several compounds known to inhibit purine synthesis de novo was studied. Adenine, orotic acid and azaserine inhibited purine synthesis, but hypoxanthine and allopurinol did not. Platelet content of phosphoribosylpyrophosphate (PRPP) and of ribose-5-pes. Incubation of intact platelets with high inorganic phosphate concentrations caused an increase in platelet PRPP content but did not affect R-5-P content or the rate of purine synthesis de novo.

Differential effects of tumor promoters on cAMP production: inhibition of receptor-mediated and potentiation of cholera toxin-mediated stimulation.

Tetradecanoylphorbol-acetate and other tumor promoters inhibit prostaglandin E2 and isoproterenol-induced cAMP accumulation in mouse thymocytes but markedly potentiate cAMP production induced by cholera toxin. Cholera toxin is known to stimulate cAMP production by inducing ADP-ribosylation of the alpha-subunit of a guanine nucleotide-binding regulatory (G) protein, resulting in activation of the catalytic unit of adenylate cyclase. G proteins have been implicated as plasma membrane transducers for a variety of additional signals. It is possible that the growth promoting and co-mitogenic properties of tumor promoters are related to their effects on G proteins.

Biochemical tissue markers of human colorectal carcinoma.

The potential therapeutic effects of differentiating agents on leukemic and solid tumor cells are being evaluated worldwide. These effects can be followed by morphologic as well as biochemical parameters. The enzymatic profile of four enzymes and the level of carcinoembryonic antigen were studied in 24 human colorectal carcinoma specimens and their adjacent uninvolved mucosa. The enzymes studied were thymidine kinase and 6-phosphogluconate dehydrogenase as markers of proliferation, and alkaline phosphatase and gamma-glutamyl transpeptidase as markers of differentiation. A consistent finding was a marked increase in the activities of thymidine kinase and 6-phosphogluconate dehydrogenase in the tumor cells as compared with the adjacent normal mucosa. The activity of gamma-glutamyl transpeptidase was not significantly different between tumor and uninvolved colon tissue. Alkaline phosphatase activity was markedly reduced in the tumor specimens. A relationship between the degree of differentiation and the degree of penetration and CEA expression was demonstrated in the tumor specimens as well as in their surrounding uninvolved mucosa.

Mitogenic and comitogenic properties of the indole alkaloid class of tumor promotors.

We determined the mitogenic and comitogenic properties of tumor promoters in the indole alkaloid series; agents that differ structurally from 12-0-tetradecanoylphorbol-13-acetate (TPA), which is known to be a lymphocyte mitogen. Teleocidin and dihydroteleocidin B were mitogenic for human lymphocytes, and lyngbyatoxin A elicited little or no mitogenesis. Catalase enhanced the mitogenicity of teleocidin and dihydroteleocidin B, and lyngbyatoxin A was also mitogenic in the presence of catalase. The potency of the agents was TPA = teleocidin greater than dihydroteleocidin B greater than lyngbyatoxin A. Dimethylsulfoxide and butyric acid markedly inhibited proliferation induced by these agents in human lymphocytes. The indole alkaloid tumor promoters were all comitogenic for murine thymocytes and induced production of interleukin-2. While the comitogenic effect of teleocidin was similar to that of TPA in its susceptibility to inhibition by dimethylsulfoxide and butyric acid, the comitogenic effect of dihydroteleocidin B and lyngbyatoxin A were less susceptible to these agents. These findings may facilitate the identification of cellular sites responsible for the inhibitory effect of differentiating agents on proliferative responses of lymphocytes.

Agents that increase cellular cAMP inhibit production of interleukin-2, but not its activity.

Proliferation of lectin-treated mouse thymocytes induced by the tumor promoter, 12-0-tetradecanoyl-13-acetate, is markedly inhibited by cAMP analogues, prostaglandin E and methylisobutylxanthine. Proliferation induced by interleukin-2 is resistant to inhibition by these compounds. The tumor promoter induces interleukin-2 production in a subpopulation of lectin-treated thymocytes, and production of this lymphokine is inhibited by agents that increase cellular cAMP.

Late administration of a lipophilic tyrosine kinase inhibitor prevents lipopolysaccharide and Escherichia coli-induced lethal toxicity.

Septic shock induced by gram-negative bacteria results primarily from excessive stimulation by lipopolysaccharide (LPS) of macrophages to produce tumor necrosis factor (TNF)-alpha and interleukin (IL)-1. The cellular effects of LPS, TNF-alpha, and IL-1 are mediated via tyrosine phosphorylation pathways. A recent report indicated that selective inhibitors of tyrosine kinases, tyrphostins of the AG126 family, protect mice against LPS-induced lethal toxicity in mice. Protection was most effective when the tyrphostin was injected before the LPS. In the present study, tyrphostin AG556, which is more lipophilic than those of the AG126 family, was effective in preventing LPS-induced lethal toxicity when administered 2 h after LPS. AG556 also prevented viable Escherichia coli-induced lethal toxicity when given 2 h before and, to a lesser extent, 2 h after the bacterial inoculation. AG556 may block a critical step downstream of the signaling pathway induced by LPS after TNF-alpha production.