ABSTRACT
The N-terminal amino acid sequence of the anti-neoplastic urinary protein (ANUP), a unique cytokine present in human granulocytes, was determined to be: Pyroglu-Leu-Lys-X-Tyr-Thr-X-Lys-Glu-Pro-Met-Thr-Ser(Thr)-Ala-Ala... This sequence showed no significant homology with any other protein when used in database searches. Furthermore, a synthetic nonapeptide corresponding to the first nine residues, with Cys in positions 4 and 7, was found to be a biologically active in vitro anti-tumour agent. An alternate method for the purification of ANUP to that previously reported is also presented. This method involves differential Amicon Diaflo membrane filtration.
Subject(s)
Antigens, Ly , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Cytokines/blood , Granulocytes/physiology , Peptide Fragments/chemistry , Peptide Fragments/pharmacology , Proteins/chemistry , Proteins/toxicity , Urokinase-Type Plasminogen Activator , Amino Acid Sequence , Cell Survival/drug effects , Granulocytes/drug effects , HeLa Cells , Humans , Leucyl Aminopeptidase , Male , Molecular Sequence Data , Peptide Fragments/toxicity , Proteins/isolation & purification , Pyroglutamyl-Peptidase I , Sequence Homology, Amino Acid , Tumor Cells, Cultured , UltrafiltrationABSTRACT
Human pancreatic ductal adenocarcinoma Capan-2, derived from a 56-yr-old male Caucasian, has been studied in both tissue culture and the nude mouse. In tissue culture, tumor cells showed epithelial-like features, whereas in the nude mouse, the tumor grew as a well-differentiated adenocarcinoma, resembling histopathologically the original neoplasm. Ultrastructurally, the neoplastic cells showed characteristics of ductal epithelium. The allozyme phenotypic profile of Tumor Capan-2 was determined in eight genetically determined loci, and chromosome studies showed a hypotetraploid pattern with a number of morphological and numerical changes. Carcinoembryonic antigen was produced in trace amounts, and lactate dehydrogenase was represented only by Isoenzyme 5, regardless of environmental conditions. The characteristics of Capan-2 tumor make it a valuable addition to the small number of available pancreatic tumor lines in studies aiming at clarifying certain aspects of the biology of this type of malignancy.
Subject(s)
Carcinoma, Intraductal, Noninfiltrating/pathology , Pancreatic Neoplasms/pathology , Animals , Carcinoma, Intraductal, Noninfiltrating/physiopathology , Cell Division , Cell Line , Enzymes/analysis , Epithelium/pathology , Humans , Isoenzymes , Karyotyping , L-Lactate Dehydrogenase/metabolism , Mice , Mice, Nude , Microscopy, Electron , Neoplasm Transplantation , Pancreatic Neoplasms/physiopathologyABSTRACT
A fraction has been isolated from human urine which exhibits antiproliferative activity against human tumour cell lines without affecting the growth of several normal diploid cell lines or tumour cells of mouse or hamster origin. The major protein present in this fraction has been characterized and tentatively designated antineoplastic urinary protein (ANUP). An S020,W value of 3.69 S was obtained by sedimentation velocity analysis, and a subunit molecular mass of 16 300 Da was obtained by sedimentation equilibrium and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Centrifugation data also indicated that the protein self-associates. The amino acid analysis of ANUP was consistent with its low pI (4.2) as determined by chromatofocusing analysis. Furthermore, the amino acid composition exhibited some features similar to collagen, as shown by high levels of proline and glycine, the absence of cysteine, and the presence of low levels of hydroxyproline.
Subject(s)
Antigens, Ly , Antineoplastic Agents/urine , Proteins/isolation & purification , Proteinuria/metabolism , Urokinase-Type Plasminogen Activator , Amino Acids/analysis , Antineoplastic Agents/pharmacology , Cell Line , Centrifugation, Density Gradient , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Humans , Proteins/pharmacology , SolubilityABSTRACT
A new tumor line derived from a human pancreatic ductal adenocarcinoma of a 56-year-old Caucasian male was established in tissue culture and the nude mouse. In tissue culture, the neoplastic cells grew as large, epithelial-like, mucin-producing cells. Injection s.c. of 1 X 10(6) cultured neoplastic cells into nude mice resulted in tumor formation histologically closely resembling the original neoplasm. Ultrastructurally, the neoplastic cells showed characteristics of ductal epithelium. The allozyme phenotypic profile of the line was established in 14 genetically determined loci, and chromosome studies showed a near-tetraploid pattern. Production of macromolecules such as lactate dehydrogenase and carcinoembryonic antigen were present in measurable amounts in culture media, tumor cell extracts, nude mouse-grown tumors, and the serum of tumor-bearing mice in amounts relative to tumor size. Pancreatic enzymes were not detected. These characteristics make tumor line SW-1990 a valuable tool in studying various aspects of the biology of human pancreatic cancer.
Subject(s)
Adenocarcinoma/pathology , Pancreatic Neoplasms/pathology , Adenocarcinoma/immunology , Animals , Carcinoembryonic Antigen/analysis , Cell Division , Cell Line , Culture Techniques/methods , Humans , Karyotyping , Kinetics , L-Lactate Dehydrogenase/analysis , Mice , Mice, Nude , Neoplasm Transplantation , Pancreatic Neoplasms/immunology , Transplantation, HeterologousABSTRACT
1. [3H]Benzo(a)pyrene and 6-substituted derivatives of [3H]benzo(a)pyrene are covalently bound to calf thymus DNA upon reaction with microsomal preparations from rats pretreated with 3-methylcholanthrene in the presence of NADPH. Two different types of cytochrome P-450-448 inhibitors, alpha-naphthoflavone and 1-benzylimidazole, show greater than 80% inhibition of the binding of benzo(a)pyrene to DNA. 2. In the presence of these inhibitors, 6-hydroxymethylbenzo(a)pyrene, 6-methylbenzo(a)pyrene and 6-formylbenzo(a)pyrene show varying degrees of inhibition of binding to DNA depending upon the inhibitor employed. 3. Polyguanylic acid is the most effective substrate for the binding of each activated polynuclear aromatic hydrocarbon; polyadenylic acid and DNA show essentially equivalent binding.
Subject(s)
Benzopyrenes/metabolism , Cytochrome P-450 Enzyme Inhibitors , Cytochromes/antagonists & inhibitors , DNA/metabolism , Microsomes/metabolism , Animals , Female , In Vitro Techniques , Lung/ultrastructure , Microsomes, Liver/metabolism , NADP/metabolism , Poly A/metabolism , Poly G/metabolism , RatsSubject(s)
Benzopyrenes/metabolism , Transferases/metabolism , Vitamin K/pharmacology , Animals , Apoenzymes/metabolism , Cytosol/enzymology , Kinetics , Lung/enzymology , Mass Spectrometry , NADP , RatsABSTRACT
alpha-Naphthoflavone activates the aryl hydroxymethyl synthetase of both the microsomal membrane-bound and soluble enzymes of rat liver and rat lung. The enzyme catalyzes the hydroxymethylation of benzo(alpha)pyrene to the 6-hydroxymethyl derivative.
