Nerve growth factor-induced alteration in the response of PC12 pheochromocytoma cells to epidermal growth factor.

PC12 cells, which differentiate morphologically and biochemically into sympathetic neruonlike cells in response to nerve growth fact, also respond to epidermal growth factor. The response to epidermal growth factor is similar in certain respects to the response to nerve growth fact. Both peptides produce rapid increases in cellular adhesion and 2-deoxyglucose uptake and both induce ornithine decarboxylase. But nerve growth factor causes a decreased cell proliferation and a marked hypertrophy of the cells. In contrast, epidermal growth factor enhances cell proliferation and does not cause hypertrophy. Nerve growth factor induces the formation of neuritis; epidermal growth factor does not. When both factors are presented simultaneously, the cells form neurites. Furthermore, the biological response to epidermal growth fact, as exemplified by the induction of ornithine decarboxylase, is attenuated by prior treatment of the cells with nerve growth factor. PC12 cells have epidermal growth factor receptors. The binding of epidermal growth factor to these receptors is rapid and specific, and exhibits an equilibrium constant of 1.9 x 10(-9) M. Approximately 80,000 receptors are present per cell, and this number is independent of cell density. Treatment of the cells with nerve growth factor reduces the amount of epidermal growth factor binding by at least 80 percent. The decrease in receptor binding begins after approximately 12-18 h of nerve growth factor treatment and is complete within 3 d. Scratchard plots indicate that the number of binding sites decreases, not the affinity of the binding sites for epidermal growth factor.

Pharmacodynamics and pharmacokinetics of the novel thrombopoietin mimetic peptide RWJ-800088 in humans.

RWJ-800088 is a novel thrombopoietin mimetic peptide for the treatment of thrombocytopenia. The objectives of this study were to evaluate the pharmacokinetics, pharmacodynamics, and safety of ascending doses of RWJ-800088 administered as a single intravenous delivery in a double-blind, placebo-controlled study with five parallel groups of eight healthy human subjects each. Platelet counts and functionality, peripheral stem cells, drug concentrations, and routine laboratory parameters were measured frequently up to day 29, and antibody formation was measured up to days 56-72. At doses > or = 0.75 microg/kg of RWJ-800088, platelet levels showed dose-related elevation as compared to results with placebo. The pharmacokinetic profile was characterized for doses of 2.5 and 3.0 microg/kg, although the dose relationship could not be fully defined. The two highest doses of RWJ-800088 appeared to increase burst-forming units-erythroid and colony-forming unit counts, suggesting some effects on progenitor lineages. RWJ-800088 was well tolerated, with no evidence of antibody formation in this single-dose study. Additional patient studies are warranted to investigate the therapeutic use of this novel peptide.

Characterization of the antitumor effects of the selective farnesyl protein transferase inhibitor R115777 in vivo and in vitro.

R115777 [(B)-6-[amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)-methyl]-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone] is a potent and selective inhibitor of farnesyl protein transferase with significant antitumor effects in vivo subsequent to oral administration in mice. In vitro, using isolated human farnesyl protein transferase, R115777 competitively inhibited the farnesylation of lamin B and K-RasB peptide substrates, with IC50s of 0.86 nM and 7.9 nM, respectively. In a panel of 53 human tumor cell lines tested for growth inhibition, approximately 75% were found to be sensitive to R115777. The majority of sensitive cell lines had a wild-type ras gene. Tumor cell lines bearing H-ras or N-ras mutations were among the most sensitive of the cell lines tested, with responses observed at nanomolar concentrations of R115777. Tumor cell lines bearing mutant K-ras genes required higher concentrations for inhibition of cell growth, with 50% of the cell lines resistant to R115777 up to concentrations of 500 nM. Inhibition of H-Ras, N-Ras, and lamin B protein processing was observed at concentrations of R115777 that inhibited cell proliferation. However, inhibition of K-RasB protein-processing could not be detected. Oral administration b.i.d. of R115777 to nude mice bearing s.c. tumors at doses ranging from 6.25-100 mg/kg inhibited the growth of tumors bearing mutant H-ras, mutant K-ras, and wild-type ras genes. Histological evaluations revealed heterogeneity in tumor responses to R115777. In LoVo human colon tumors, treatment with R115777 produced a prominent antiangiogenic response. In CAPAN-2 human pancreatic tumors, an antiproilferative response predominated, whereas in C32 human melanoma, marked induction of apoptosis was observed. The heterogeneity of histological changes associated with antitumor effects suggested that R115777, and possibly farnesyl protein transferase inhibitors as a class, alter processes of transformation related to tumor-host interactions in addition to inhibiting tumor-cell proliferation.

On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells.

The time course of corticotropin-induced steroidogenesis and changes in intracellular cyclic AMP and cyclic GMP levels were investigated in isolated bovine adrenocortical cells prepared by trypsin digestion. Corticotropin produced a peak rise in cyclic AMP during the first 5 min of stimulation and enhanced steroid production after 15 min. Corticotropin also caused a decrease in cortical cyclic GMP at 5 min; this decrease in cyclic GMP reverted to a 2-3 fold increase at 15-30 min which gradually subsided by 60 min. A steroidogenic concentration of prostaglandin E2 also produced an elevation in the levels of both nucleotides, but the rise in cyclic GMP preceded the rise in cyclic AMP. These results suggest that the relative amounts of cyclic AMP and cyclic GMP, rather than the absolute levels of cyclic AMP, may be a key factor in the regulation of steroidogenesis.

Phase I and pharmacokinetic study of farnesyl protein transferase inhibitor R115777 in advanced cancer.

PURPOSE: To determine the maximum-tolerated dose, toxicities, and pharmacokinetic profile of the farnesyl protein transferase inhibitor R115777 when administered orally bid for 5 days every 2 weeks. PATIENTS AND METHODS: Twenty-seven patients with a median age of 58 years received 85 cycles of R115777 using an intrapatient and interpatient dose escalation schema. Drug was administered orally at escalating doses as a solution (25 to 850 mg bid) or as pellet capsules (500 to 1300 mg bid). Pharmacokinetics were assessed after the first dose and the last dose administered during cycle 1. RESULTS: Dose-limiting toxicity of grade 3 neuropathy was observed in one patient and grade 2 fatigue (decrease in two performance status levels) was seen in four of six patients treated with 1,300 mg bid. The most frequent clinical grade 2 or 3 adverse events in any cycle included nausea, vomiting, headache, fatigue, anemia, and hypotension. Myelosuppression was mild and infrequent. Peak plasma concentrations of R115777 were achieved within 0.5 to 4 hours after oral drug administration. The elimination of R115777 from plasma was biphasic, with sequential half-lives of about 5 hours and 16 hours. There was little drug accumulation after bid dosing, and steady-state concentrations were achieved within 2 to 3 days. The pharmacokinetics were dose proportional in the 25 to 325 mg/dose range for the oral solution. Urinary excretion of unchanged R115777 was less than 0.1% of the oral dose. One patient with metastatic colon cancer treated at the 500-mg bid dose had a 46% decrease in carcinoembryonic antigen levels, improvement in cough, and radiographically stable disease for 5 months. CONCLUSION: R115777 is bioavailable after oral administration and has an acceptable toxicity profile. Based upon pharmacokinetic data, the recommended dose for phase II trials is 500 mg orally bid (total daily dose, 1, 000 mg) for 5 consecutive days followed by 9 days of rest. Studies of continuous dosing and studies of R115777 in combination with chemotherapy are ongoing.

Preclinical antitumor activity and pharmacodynamic studies with the farnesyl protein transferase inhibitor R115777 in human breast cancer.

Antitumor and pharmacodynamic studies were performed in MCF-7 human breast cancer cells and companion xenografts with the farnesyl protein transferase inhibitor, R115777, presently undergoing Phase II clinical trials, including in breast cancer. R115777 inhibited growth of MCF-7 cells in vitro with an IC(50) of 0.31 +/- 0.25 microM. Exposure of MCF-7 cells to increasing concentrations of R115777 for 24 h resulted in the inhibition of protein farnesylation, as indicated by the appearance of prelamin A at concentrations >1 microM. After continuous exposure to 2 microM R115777, prelamin A levels peaked at 2 h post drug exposure and remained high for up to 72 h. R115777 administered p.o. twice daily for 10 consecutive days to mice bearing established s.c. MCF-7 xenografts induced tumor inhibition at a dose of 25 mg/kg [percentage of treated versus control (% T/C) = 63% at day 21]. Greater inhibition was observed at doses of 50 mg/kg (% T/C at day 21 = 38%) or 100 mg/kg (% T/C at day 21 = 43%). The antitumor effect appeared to be mainly cytostatic with little evidence of tumor shrinkage to less than the starting volume. Tumor response correlated with an increase in the appearance of prelamin A, but no changes in the prenylation of lamin B, heat shock protein 40, or N-Ras were detectable. In addition, significant increases in apoptotic index and p21(WAF1/CIP1) expression were observed, concomitant with a decrease in proliferation as measured by Ki-67 staining. An increase in prelamin A was also observed in peripheral blood lymphocytes in a breast cancer patient who responded to R115777. These data show that R115777 possesses preclinical antitumor activity against human breast cancer and that the appearance of prelamin A may provide a sensitive and convenient pharmacodynamic marker of inhibition of prenylation and/or response.

Experimental studies with liarozole (R 75,251): an antitumoral agent which inhibits retinoic acid breakdown.

Liarozole reduced tumor growth in the androgen-dependent Dunning-G and the androgen-independent Dunning MatLu rat prostate carcinoma models as well as in patients with metastatic prostate cancer who had relapsed after orchiectomy. In vitro, liarozole did not have cytostatic properties, as measured by cell proliferation in breast MCF-7 and prostate DU145 and LNCaP carcinoma cell lines. It did not alter the metabolism of labeled testosterone i.e. the 5 alpha-reductase in cultured rat prostatic cells. In mouse F9 teratocarcinoma cells liarozole did not show any retinoid-like properties but enhanced the plasminogen activator production induced by retinoic acid. Furthermore, liarozole and retinoic acid similarly reduced the growth of the androgen-dependent Dunning-G tumor in nude mice and inhibited tumor promotion elicited by phorbol ester in mouse skin. These data have raised the hypothesis that the antitumoral properties of liarozole may be related to inhibition of retinoic acid degradation, catalyzed by a P-450-dependent enzyme that is blocked by the drug.

Identification of nerve growth factor receptors in primary cultures of chick neural crest cells.

Primary cultures of chick neural crest cells obtained from explanted neural tubes have binding sites for radioiodinated nerve growth factor ([125I]NGF) but not for radioiodinated epidermal growth factor ([125I]EGF). The binding of [125I]NGF was shown to be a specific and saturable process with a high affinity (Kd = 0.3 nM) for the ligand. Despite the expression of these NGF binding sites, incubation of the neural crest cultures with nerve growth factor did not induce neurite outgrowth; no morphological alterations were observed. This was not due to an inability of the cells to express a neuronal phenotype, since the neural crest cells spontaneously differentiated into neurite-bearing cells. However, the nerve growth factor binding sites do appear to be functional receptors, since nerve growth factor could produce a modest induction of ornithine decarboxylase. The quantity of nerve growth factor binding sites seemed to be independent of the phenotype expressed by the neural crest cells, since both pigmented cells and neuron-like neural crest cells exhibited binding. These findings suggest that the differentiation of neural crest cells into mature nerve growth factor-responsive neurons may involve the coupling of nerve growth factor receptors to cellular responses important in the expression of the neuronal phenotype.

A rapid assay for measuring the metabolism of [3H]-retinoic acid in cell cultures.

A simple method was developed to detect the metabolism of [3H]-retinoic acid to polar products using intact tumor cells in culture. Unaltered [3H]-retinoic acid was separated from more polar metabolites using C18-bonded solid phase extraction cartridges. Separation of unaltered retinoic acid and polar metabolites was confirmed by HPLC. The murine mammary carcinoma cell line TA3 Ha used in these studies converted 40% to 50% of added radioactive retinoic acid to polar metabolites released into the culture medium during a 4-hr incubation period. Metabolism of [3H]-retinoic acid by TA3 Ha cells was inhibited by the cytochrome P-450 inhibitors ketoconazole, clotrimazole, and liarozole. The simplicity and rapidity of this assay should make it useful for evaluating compounds as inhibitors of retinoic acid metabolism.

Inhibition of leukotriene and thromboxane biosynthesis by a 9-(4-chlorophenyl) analogue of arachidonic acid.

The synthesis of 9-(4-chlorophenyl)-7,7-dimethyl-5(Z), 8-nonadienoic acid (7) and its methyl ester 6, and their effects on arachidonic acid metabolism in vitro are described. The IC50 values of 19.6 and 20.6 microM were observed for inhibition of leukotriene synthesis in human granulocytes for 6 and 7, respectively. Additionally, the compounds inhibited thromboxane B2 (TxB2) synthesis, with respective IC50 values of 6.1 and 20 microM, while producing pronounced 3-8-fold increases in PGE2 synthesis in human mononuclear cells. Increased PGE2 synthesis may have reflected shunting of free arachidonic acid substrate at the thromboxane synthetase and endoperoxide E isomerase branchpoint of arachidonic acid metabolism.

Simultaneous visualization of the binding of nerve growth factor and epidermal growth factor to single rat pheochromocytoma (PC12) cells through indirect immunohistofluorescence.

The rat pheochromocytoma cell line, PC12, which has receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), was used to develop a technique for the simultaneous visualization of separate growth factor receptors by indirect immunohistofluorescence. The cells were incubated with saturating concentrations of nerve growth factor and epidermal growth factor. After fixation, the cells were treated with anti-NGF sheep antiserum and then with antisheep rabbit IgG conjugated with fluorescein; they also were treated with anti-EGF rabbit antiserum and then with anti-rabbit sheep IgG conjugated with rhodamine. Fluorescence microscopy showed that a single PC12 cell bound both NGF and EGF. The fluorescence due to EGF binding was reduced when the cells were grown in the presence of NGF. A similar reduction of fluorescence was observed after addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Both manipulations are known to reduce the specific binding of 125I-EGF to these cells. Subclones of PC12 cells, NR11 and NR20, reported not to have NGF receptors, did not demonstrate NGF binding when tested with this indirect immunohistofluorescence method. Thus, the binding of growth factors which is demonstrable by indirect immunohistofluorescence method seems to reflect the presence of the specific cell surface receptors for both peptides on individual PC12 cells.

Farnesyl protein transferase inhibitors and other therapies targeting the Ras signal transduction pathway.

The year 2000 will be a significant date for the field of Ras-related therapies since numerous agents will have Phase II clinical efficacy data maturing to provide proof of principle for this cancer treatment strategy. These data will also provide an important milestone for the cancer research community since these molecules represent a small vanguard of oncology drug discovery projects predicated on molecular targets. We can only hope that these agents are a successful harbinger for the formidable number of targeted therapies that will be entering development pipelines in the coming years.

Characterization of a high affinity interleukin-1 (IL-1) specific binding site in a human synovial sarcoma (Hs431) cell line.

A subpopulation of cells was derived from the Hs431 connective tissue sarcoma cell line which possessed high affinity (estimated Kd = 0.38-0.55 nM) binding sites for human recombinant [125I]-IL-1 alpha. Binding at 4 degrees C was slow approaching equilibrium by 4 hrs. Dissociation of [125I]-IL-1 alpha was also slow and unaffected by high concentrations of cold ligand. The binding site also underwent ligand-induced internalization at 37 degrees C. An Mr = 83,000 protein was identified in affinity crosslinking studies. Despite these similarities to previously reported IL-1 receptors, Hs431 cells did not exhibit biological responses to IL-1 which have been observed in other cell lines. IL-1 did not induce PGE2 or collagenase synthesis. IL-1 also failed to induce ornithine decarboxylase activity (ODC) or stimulate [3H]-thymidine incorporation. In contrast, the Hs431 cells did contain a functional epidermal growth factor (EGF) receptor as determined from binding studies, protein kinase activity, induction of ODC, and stimulation of [3H]-thymidine incorporation. Thus, the refractoriness of Hs431 cells to IL-1 was fairly specific and did not result from a generalized defect associated with cell transformation.

The induction of ornithine decarboxylase by nerve growth factor and epidermal growth factor in PC12 cells.

Both nerve growth factor and epidermal growth factor cause an induction of ornithine decarboxylase in the rat pheochromocytoma clone PC12. The induction by nerve growth factor is transcription-dependent and occurs within 4 to 6 h. Antibody studies indicate that nerve growth factor must be present for 2-3 h to obtain full induction. Nerve growth factor is synergistic with either N6, O2-dibutyryl cyclic 3',5'-adenosine monophosphate (dBcAMP) or 3-isobutyl-1-methylxanthine (IBMX) in the induction. The magnitude of ornithine decarboxylase induction is influenced by the density of the culture. Synchronized cell populations show the greatest sensitivity to nerve growth factor just before, or immediately upon, entering S phase. The induction of ornithine decarboxylase by epidermal growth factor appears to be quite similar to that exhibited by nerve growth factor. Epidermal growth factor is active in the range of ng/ml. The time course of the induction is the same, as is the need for the peptide to remain in contact with the cells for several hours. Putrescine inhibits the induction and dBcAMP and IMBX accentuate it. Cells appear to be sensitive to epidermal growth factor also near the G1/S border. In spite of the marked similarities in these inductions, a maximal level of nerve growth factor plus a maximal level of epidermal growth factor yields greater induction than either alone, indicating the inductions occur by somewhat different mechanisms.

Molecular size of the epidermal growth factor receptor-kinase as determined by radiation inactivation.

Radiation inactivation with high energy electrons from a linear accelerator was used to determine the functional molecular size of the epidermal growth factor (EGF) binding site and the tyrosine-specific protein kinase activity in A-431 membranes. The target size of the protein portion of the EGF receptor glycoprotein was 147,000 daltons when the radiation-dependent decrease in maximal binding capacity was measured. Since the target size is in good agreement with the molecular size of the protein portion of the EGF receptor determined by denaturing biochemical methods, it appears that the monomeric receptor is the functional binding site in situ. The target size of the EGF-stimulated kinase activity associated with the affinity-purified EGF receptor/kinase was 133,000 and 144,000 daltons when assayed for the ability to autophosphorylate or to phosphorylate a tyrosine-containing peptide, respectively. However, the target size of the kinase activity that did not adhere to an EGF-affinity column was 54,000 and 69,000 daltons when assayed for phosphorylation of endogenous and exogenous substrates, respectively. Intermediate target sizes were obtained when kinase assays were performed on membranes prior to fractionation by affinity chromatography. These results, taken with other biochemical data, indicate that A-431 membranes contain a kinase activity that is a domain of the glycoprotein that contains the EGF binding site and that the membranes also contain another tyrosine-specific kinase or kinases that have an average size of approximately 60,000 daltons.

Nerve growth factor-induced decrease in the cell-free phosphorylation of a soluble protein in PC12 cells.

Incubation of cell-free extracts from PC12 cells with [32P]ATP leads to the phosphorylation of a 100,000-dalton protein. In extracts from cells treated with nerve growth factor, the labeling of the 100,000-dalton protein is substantially and selectively reduced. Direct quantitation indicates that the reduction is a minimum of 30-50% in the various experiments. The decrease is evident after as little as 15 min of nerve growth factor treatment, and disappears within 2 h after the removal of nerve growth factor. The decrease is dose dependent; a complete response is seen after treatment with 10 ng of nerve growth factor/ml. Some decrease in phosphorylation is also seen after treatment of the cells with epidermal growth factor, 12-O-tetradecanoylphorbol-13-acetate, or 5'-N-ethylcarboxamideadenosine, a potent adenosine receptor agonist, but not after treatment with insulin. The phosphorylation of the 100,000-dalton protein, in extracts from either control or nerve growth factor-treated cells, leads almost exclusively to the formation of phosphothreonine. The addition of equal amounts of extract from untreated cells and extract from nerve growth factor-treated cells produces a level of phosphorylation exactly intermediate between those of the two extracts used separately, indicating the absence of a soluble kinase inhibitor. The data suggest that nerve growth factor treatment produces either a covalent inhibition or a physical removal of the kinase for the 100,000-dalton protein.

Effects of 12-0-Tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: interactions with epidermal growth factor and nerve growth factor.

The phorbol ester tumor promotor 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (125I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent Ki of 11-26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of 3H-choline and 32 P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth fact (NGF) which, in contrast to the former agents, is a differentiating stimulus for PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of 125I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.

The action of adenosine analogs on PC12 cells.

PC12 cells, a nerve growth factor-responsive clone of rat pheochromocytoma, contain a membrane-bound adenylate cyclase, which can be activated by adenosine analogs. The characteristics of the cyclase response indicate the presence of stimulatory adenosine receptors. Adenosine analogs also produce a marked increase in the ornithine decarboxylase levels of the cells, and the characteristics of this response suggest that it is linked to the adenylate cyclase-stimulatory adenosine receptors. The ornithine decarboxylase response elicited by 5'-N-ethylcarboxamideadenosine (NECA), a potent stimulatory adenosine analog, is synergistic with that produced by nerve growth factor. Differentiation of the cells with nerve growth factor, however, does not substantially alter either the response of cyclase to the adenosine analog or the magnitude of the adenosine-evoked ornithine decarboxylase response. Treatment of the cells with NECA produces an increase in the phosphorylation of a specific non-histone nuclear protein. While causing little or no morphological alteration by itself, NECA is synergistic with nerve growth factor in producing neurite outgrowth in PC12 cells. NECA does not cause an induction of acetylcholinesterase in the cells. NECA does not cause an induction of acetylcholinesterase in the cells, nor does it appear to affect the induction of this enzyme by nerve growth factor.

Neurochemical correlates of chlordecone neurotoxicity.

The neurotoxic organochlorine insecticide chlordecone (Kepone) was examined in several in vitro and in vivo neurochemical systems in an attempt to identify neurochemical alterations that might be relevant to the central nervous system manifestations of chlordecone toxicity in humans. In vitro, chlordecone was a remarkably potent inhibitor of brain mitochondrial oxidative phosphorylation and associated Ca2+ transport (Ki congruent to 10(-7) M). At a high concentration of chlordecone (10(-5) M), destabilization of biological membranes was observed. Both of these effects appeared to contribute to inhibition of synaptosomal Ca2+ uptake, which was accompanied by a pronounced, although paradoxical, stimulation of neurotransmitter release. Studies of the disposition of [14C]chlordecone revealed that the concentrations that elicited neurochemical changes in vitro were comparable to the brain tissue chlordecone concentrations achieved with a 40 mg/kg tremorigenic dose in intact animals. However, no neurochemical correlates of chlordecone toxicity were observed in studies of dopamine and norepinephrine turnover in chlordecone-intoxicated animals. These findings are discussed in relation to the development of neurochemical assays appropriate for investigating neurotoxic agents.