Differential growth sensitivity to 4-cis-hydroxy-L-proline of transformed rodent cell lines

The effect of 4-cis-hydroxy-L-proline (CHP), a proline analogue, on the anchorage-dependent and -independent growth of several transformed rodent cell lines was studied. Mouse NIH-3T3 fibroblasts transformed by a variety of different oncogenes (Ki-ras, mos, src, fms, fes, met, and trk) by a DNA tumor virus (SV40) or by a chemical carcinogen (N-methylnitrosourea) were all found to be more sensitive (50% inhibitory dose, 20 to 55 micrograms/ml) to the dose-dependent inhibitory effects of CHP on growth in monolayer culture than were NIH-3T3 cells (50% inhibitory dose, 120 micrograms/ml). CHP was generally found to be even more effective in inhibiting the growth of these transformed cells as colonies in soft agar than in monolayer cultures. In addition, rat embryo fibroblasts (CREF) and normal rat kidney fibroblasts (NRK) after transformation with a Ki-ras oncogene exhibit a similar increase in their sensitivity to CHP-induced growth inhibition. Treatment of NRK cells with transforming growth factor alpha (TGF-alpha) and beta (TGF-beta), which reversibly induces phenotypic transformation of these cells, increases their sensitivity to CHP to a level comparable with that observed in Ki-ras-transformed NRK cells (K-NRK). The growth inhibitory effects of CHP are reversible, since removal of CHP results in a normal resumption of cell growth. CHP uptake occurs primarily through the Na+- and energy-dependent neutral amino acid transport A system, which is 6- to 7-fold more elevated in K-NRK cells compared with NRK cells. Treatment of NRK cells with TGF-alpha and/or -beta increases the uptake of [3H]methylaminoisobutyric acid on the A system to a level that is similar to that found in K-NRK cells. The functions of the Na+/K+ and Na+/H+ exchange systems are apparently necessary for the enhanced A system activity, since ouabain and amiloride can inhibit the uptake of [3H]methylaminoisobutyric acid in K-NRK cells and in NRK cells treated with TGF-alpha and/or -beta. The activity of the A system is specifically increased in K-NRK and in TGF-alpha- and/or -beta-treated NRK cells, since the other two major neutral amino acid uptake systems, the ASC and the L systems, and the Ly+ system for basic amino acid uptake show no apparent changes in their activity in NRK cells after treatment with TGF-alpha and/or -beta or in these cells after transformation with the Ki-ras oncogene. These results suggest that the differential growth sensitivity to CHP of transformed rodent cells and of normal fibroblasts treated with TGF-alpha and/or -beta is due in part to an elevated uptake of this amino acid analogue on the neutral amino acid transport A system.     

Effects of ouabain on NIH/3T3 cells transformed with retroviral oncogenes and on human tumor cell lines

Both murine and human cell lines transformed by the v-Ki-ras gene have been shown to be much more sensitive to the toxic effects of the cardiac glycoside ouabain than their respective controls. This differential toxicity has previously been used in the isolation of flat revertant clones from populations of Kirsten murine sarcoma virus transformed NIH/3T3 cells. Here, we have undertaken a further characterization of this phenomenon in murine and human tumor cells. Two different techniques, a 51Cr-release assay and a quantitative Crystal violet elution assay, have been employed to compare the sensitivities to ouabain of normal and v-Ki-ras-transformed NIH/3T3 cells. In each assay, ras-transformed NIH/3T3 cell lines displayed an increased sensitivity to ouabain as compared to the parental NIH/3T3 cell line, both in dose-response and in time-course experiments. In a separate study, ouabain was also able to inhibit the growth in semi-solid medium of 2 v-Ki-ras-transformed NIH/3T3 cell lines (DT and K-NIH) in a dose-dependent fashion. The same concentrations of ouabain were effective in both the 51Cr-release and Crystal violet assays. To address the question of whether increased sensitivity to ouabain is a specific result of transformation with the ras oncogene or is a common event which accompanies transformation by other oncogenes, we have screened a variety of transformed NIH/3T3 derivatives. All of these lines displayed an increased sensitivity to ouabain when compared to the parental NIH/3T3 cell line.     

Stimulatory effects of brain natriuretic peptide on cyclic GMP accumulation and tyrosine hydroxylase activity in cultured bovine adrenal medullary cells

Summary We studied the effect of brain natriuretic peptide (BNP) on the accumulation of cyclic GMP and the phosphorylation and activity of tyrosine hydroxylase, compared with that of atrial natriuretic peptide (ANP), in cultured bovine adrenal medullary cells. 1. BNP as well as ANP increased cellular cyclic GMP accumulation in a concentration-dependent manner (10–1000 nmol/1). BNP (1 mol/1) and ANP (1 mol/1) produced a 60-fold and 30-fold increase in cyclic GMP accumulation, respectively. 2. The stimulatory effects of BNP and ANP on cyclic GMP accumulation were observed even when Ca²⁺ or Na⁺ was removed from the incubation medium. 3. 12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of protein kinase C, inhibited the stimulatory effect of BNP on cyclic GMP accumulation in a concentration-dependent manner (1–100 nmol/1). Furthermore, the BNP-induced accumulation of cyclic GMP was attenuated by forskolin (1 mol/1), an activator of adenylate cyclase. 4. BNP (1 mol/1) and ANP (1 mol/1) caused a significant increase in phosphorylation and activity of tyrosine hydroxylase in the cells. 5. In digitonin-permeabilized cells, cyclic GMP (1–100 mol/1) activated tyrosine hydroxylase in the presence of ATP and Mg²⁺.These results suggest that BNP stimulates the accumulation of cyclic GMP in a manner similar to that of ANP. The increased accumulation of cyclic GMP by these peptides may be negatively modulated by protein kinase C and cyclic AMP and may cause the phosphorylation and activation of tyrosine hydroxylase-in cultured bovine adrenal medullary cells.     

Expression of a novel matrix metalloproteinase regulator, RECK, and its clinical significance in resected non-small cell lung cancer

The reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) was initially isolated as a transformation-suppressor gene by expression cloning and found to encode a membrane-anchored regulator of the matrix metalloproteinases (MMPs). Experimental studies have shown that RECK can suppress tumour - invasion, metastasis and angiogenesis. However, the clinical impact of RECK remains unclear. To assess the clinical significance of RECK-expression in non-small cell lung cancer (NSCLC), a total of 171 patients with completely resected pathological stage (p-stage) I-IIIA NSCLC were retrospectively examined. Expression of RECK and vascular endothelial growth factor (VEGF) in tumour tissues was assessed by immunohistochemical staining (IHS). Intratumoural microvessel density (IMVD), a measurement of angiogenesis, was also determined by IHS using an anti-CD34 antibody. A significant inverse correlation between RECK-expression and tumour angiogenesis was documented; the mean IMVD in tumours with strong RECK-expression (157.1) was significantly lower than that observed in tumours with weak RECK-expression (194.5; P = 0.008). Interestingly, this inverse correlation was seen only when VEGF was strongly expressed, which suggests that RECK could suppress the angiogenesis induced by VEGF. The 5-year survival rate for patients with tumours with strong RECK-expression (75.8%) was significantly higher than that for patients with weakly expressing tumours (54.3%; P = 0.016). Subset analyses showed that the prognostic impact of RECK-status was evident in patients with either adenocarcinoma, poorly differentiated tumours, or p-stage IIIA disease. A multivariate analysis confirmed that reduced RECK-expression was an independent and significant factor in predicting a poor prognosis (P = 0.009; Hazard ratio (HR), 0.474 with a 95% Confidence interval (CI) of 0.271-0.830). In conclusion, RECK-status is a significant prognostic factor correlated with tumour angiogenesis in NSCLC patients.     

Generation of lymphomagenic mouse type-C viruses from radiation- or chemically-induced non-producer T-cell lymphoma cell lines infected with non-oncogenic ecotropic virus

Highly lymphomagenic mouse type-C viruses were generated from radiation- or chemically-induced T-cell lymphoma cell lines of NFS/N mouse origin infected with a non-oncogenic ecotropic virus E4. By analysis of these progeny viruses, the following results were obtained. 1) The viruses were lymphomagenic in neonatally inoculated NFS/N and C3H/He mice and W/Fu rats but not in Balb/c and C57BL/6N mice, indicating that they possess the Fv-1n tropism of exogenously infected parent virus. 2) Lymphomagenic viruses consisted of plural viral subpopulations. Recombinant mink cell focus-inducing (MCF) and ecotropic viruses were cloned from them. Inoculation of either MCF or ecotropic virus alone or both viruses together did not cause lymphoma in NFS/N mice and there was no evidence of viral replication in the recipients. 3) Inoculation of either MCF- or ecotropic virus-infected NFS-ME cells alone did not cause lymphoma development in pre-irradiated NFS/N mice, while transplantation of both MCF- and ecotropic virus-infected NFS-ME cells resulted in the development of lymphomas of host origin. These results show that lymphomagenic MCF virus was generated through the recombination of E4 viral genome and a modified proviral DNA of endogenous viruses present in radiation- or chemically-induced lymphomas, and that an interaction or synergism of MCF and ecotropic viruses is required for MCF virus to exert lymphomagenic activity.     

Tubulovesicular structures in experimental Creutzfeldt-Jakob disease and scrapie

Tubulovesicular structures, measuring 20-50 nm in diameter, were found in dilated neuronal processes in brains from mice infected with the Fujisaki strain of Creutzfeldt-Jakob disease virus. These particles were similar to those observed in brains from hamsters infected with scrapie. These structures are consistently present in naturally occurring and experimentally induced spongiform encephalopathies, irrespective of the host species or virus strain. Their role in pathogenesis is undetermined.     

Antidromic activation of vagal and sympathetic afferents does not produce intestinal contractions in dogs.

The question has been asked whether vagal and sympathetic afferents activated antidromically play a role as motor nerves on the in vivo small intestine in dogs anesthetized with urethane. The vagus nerve of one side was cut above the nodose ganglion and the efferent fibers allowed to degenerate. Peripheral stimulation (5-50 Hz, 0.5-3 ms, 5-25 V) of an intact cervical vagus, being able to excite both efferent and afferent fibers, caused large contractions in the jejunum and stomach, whereas stimulation of the contralateral cut cervical vagus could not produce any response in the jejunum but small contractions in the stomach. Peripheral stimulation of the cut cervical vagus did not produce bradycardia and hypotension. Single- and multi-unit discharges to distension of the jejunal segments could be recorded from the peripheral cut end of the cut cervical vagus. Immunohistochemically, there were many substance P-containing cells in both nodose ganglia. Antidromic stimulation of the dorsal roots (T7-T10) did not induce any response in the jejunum but contractions in the stomach. The results may confirm that vagal and sympathetic afferents have no antidromic motor function at least in the in vivo canine small intestine.     

Histological investigation of the myenteric plexus of the entire gut in an infant with hypogenesis of the intestine.

A male infant with pseudo-Hirschsprung's disease was treated for 2 years and 10 months, then died of severe enterocolitis. At autopsy examination of the entire gut was possible, and a definite histological examination could be performed which threw light on the pathogenesis. The ganglion cells of the esophagus, stomach, small intestine, colon and rectum were examined, diameters of their nuclei were measured, and their nuclear volumes were calculated. S-100 and anti-neurofilament stainings were also performed in an immunohistological investigation of the glial cells and nerve fibers. Both the diameter of the volume of the nuclei of the ganglion cells in the small intestine and colon were significantly smaller than normal. Even in the esophagus the nuclear volume was smaller than normal. The glial cells and nerve fibers gradually decreased in the caudal direction. Thus, histomorphometry and immunohistochemistry both showed that the neuroblasts in the intestine were immature and the migration of ganglion cells was disturbed in this case. The final diagnosis was hypogenesis of the intestine.     

Anti-cephalexin monoclonal antibodies and their cross-reactivities to cephems and penams

Three cell lines producing monoclonal antibodies (MAbs), Cep1-2, 2-2 and 6, against cephalexin were established and the immunoglobulin class of the MAbs was IgM. The cross-reactions of the MAbs with penams and cephems were examined by enzyme-linked immunosorbent assay (ELISA). The cross-reactivities of Cep1-2 and 2-2 were scarcely influenced by the structures of acyl side chains of cephems and penams. The cross-reactivities of Cep1-2 were affected by the presence or absence of dihydrothiazolidine ring of cephem nucleus in hapten-protein conjugates which were prepared by alkaline method, MBS method and activated ester method but the cross-reactivities of Cep2-2 were not. The findings suggest that Cep1-2 recognize the degradate product(s) of cephem nucleus and Cep2-2 recognize a new antigenic determinant (NAD), which is formed by the conjugation of beta-lactam and carrier protein. On the other hand, the cross-reactivities of Cep6 were influenced by the structure of amino acyl side chain. It seems that Cep6 recognize specifically the acyl side chain at the C-7 of cephem. In ELISA inhibition test, three MAbs showed different inhibition pattern. The reaction of Cep1-2 with cephalexin-HSA was inhibited by cephalexin lysate. Cep2-2 and Cep6 were weakly inhibited by the binding to cephalexin-HSA by cephalexin lysate. Furthermore, the reactions of all MAbs were remarkably inhibited by penicillamine. The above results indicate that the MAbs can recognize at least three epitopes of the degradate product(s) of cephem nucleus, NAD and acyl side chain in cephalexin-protein conjugate.