Establishment of a new myeloid leukemia cell line (TK-1), and isolation of cells having a translocation involving a band 17q23

A myeloid leukemia cell line (TK-1) was established from the peripheral blood of a patient with lymphoblastic lymphoma whose leukemia cells were composed of T-lymphoblasts and immature myeloid cells. The established TK-1 cell line consisted of immature myeloid cells with heavy azurophilic granulation in the cytoplasm. The TK-1 cells were positive for peroxidase, and exhibited a strong positive reaction for alpha-naphthyl acetate esterase and naphthol AS-D chloroacetate esterase. The cells were weakly positive for Fc gamma-receptors, showed no phagocytosis and did not reduce NBT. With the treatment of 1 alpha, 25-dihydroxy-vitamin D3, they exhibited morphological and functional differentiation. The TK-1 cell line contained normal diploid cells and pseudodiploid cells, and the two populations were successfully cloned; the clone with a normal karyotype was designated the TK-1D cell line, and the clone with a pseudodiploid karyotype, which had a translocation involving chromosomes 14, 17 and one other chromosome, was designated the TK-1B cell line. These cloned cells lacked Epstein-Barr virus nuclear antigens and had almost the same myelomonocytic characteristics as the parent TK-1 cells. The breakpoint of chromosome 17 involved in the translocation of the pseudodiploid cells was identified to be a band 17q23.     

Gangliosides and sialoglycoproteins carrying a rare blood group antigen determinant, Cad, associated with human cancers as detected by specific monoclonal antibodies

Two murine monoclonal antibodies, 2A3D2 and 2D11E2 (both IgM), which are directed to the gangliosides and sialoglycoproteins related to a rare blood group antigen, Cad, were obtained by using a ganglioside mixture prepared from human hepatocellular carcinoma cells (PLC/PRF/5) as the immunogen. These two monoclonal antibodies detected multiple ganglioside antigens present in the PLC/PRF/5 cells, and the major antigenic ganglioside was characterized as IV4GalNAc beta-GD1a, which has the carbohydrate structure GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3GalNAc beta 1---- 4(NeuAc alpha 2----3)Gal beta 1----Cer. The two antibodies also reacted with GM2 (GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer) and a Cad-active lactoseries ganglioside (IV4GalNAc beta-sialosylparagloboside, GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4GlcNAc beta 1---- 3Gal beta 1----4Glc beta 1----Cer), which have carbohydrate structures related to IV4GalNAc beta-GD1a. Beside gangliosides, both antibodies recognized the carbohydrate determinant carried by glycophorin A on very rare Cad-positive human RBC; the structure of which is GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----3(NeuAc alpha 2---- 6)GalNAc alpha 1----Ser/Thr. From these findings, it is clear that monoclonal antibodies 2A3D2 and 2D11E2 both recognize the nonreduced carbohydrate terminus composed of three sugar residues, GalNac beta 1----4(NeuAc alpha 2----3)Gal beta 1----R, and are useful for detecting the Cad-related antigen in cells and tissues. By using these monoclonal antibodies, it was revealed that many cultured human hepatocellular carcinoma cell lines and cancer tissues taken from patients with hepatocellular carcinoma contain both Cad-active glycoprotein antigens and related gangliosides, while normal liver tissues contain no appreciable amount of either species of antigen. The Cad-active glycoprotein antigens in cultured human hepatocellular carcinoma cells appeared as triplet bands having molecular weights of 92,000, 75,000, and 61,000, under either reducing or nonreducing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Essentially the same triplet proteins were observed in as many as 4 of 9 cases (44%) of cancer tissue from patients with hepatocellular carcinoma, but not in neighboring cirrhotic tissues or normal livers tissues. These results suggest that the rare blood group antigen Cad is associated with human cancers, especially hepatocellular carcinoma.     

Serine/threonine kinase, Cot/Tpl2, regulates renal cell apoptosis in ischaemia/reperfusion injury

Aim: Cot/Tpl2, a serine/threonine (Ser/Thr) protein kinase, has been classified as a member of the mitogen‐activated protein kinase (MAPK) family, and is known to have a pleiotropic role. Many studies have reported the involvement of Cot/Tpl2, mainly as a member of the Toll‐like receptor (TLR) 4 signalling pathway in lipopolysaccharide (LPS)‐induced tumor necrosis factor‐α (TNF‐α) production. At the same time, it is also related to the caspase‐dependent apoptotic pathway. Thus, the role of Cot/Tpl2 in ischaemia/reperfusion injury (IRI) in which TNF‐α and apoptosis are the major pathogenetic factors was studied. Methods: IRI was induced in wild type (Cot/Tpl2^+/+) mice and in Cot/Tpl2‐deficient (Cot/Tpl2^?/?) mice. The extent of tubular injury and renal function were studied. TNF‐α production, neutrophil infiltration and apoptosis were also compared between the two groups. Results: Cot/Tpl2^?/? mice had preserved renal function compared with wild type mice in IRI. Although Cot/Tpl2 was phosphorylated in IRI and in the cultured tubular epithelial cells (TEC) after stimulation with LPS and hydrogen peroxide, there were no significant differences in terms of TNF‐α production, neutrophil infiltration or MAPK activation between Cot/Tpl2^+/+ and Cot/Tpl2^?/? mice. In contrast, Cot/Tpl2^?/? mice showed obviously reduced terminal deoxynucleotidyl transferase‐mediated dUTP nick end labelling positive cells and cleaved caspase‐3 positive cells. Furthermore, Cot/Tpl2‐deficient TECs demonstrated significantly less caspase‐3 activation after hydrogen peroxide stimulation with comparable caspase‐9 activation to wild type TEC. Conclusion: Cot/Tpl2 did not function as a member of MAPK family, but as a promoter of apoptosis in IRI. These results suggest that Cot/Tpl2 could be a possible therapeutic target in IRI.     

l-Carnitine mediates protection against DNA damage in lymphocytes of aged rats

It has been proposed that age-associated disorders are related to a time-dependent shift in the antioxidant/prooxidant balance towards oxidative damage. Increased production of oxidants in vivo can cause damage to intracellular macromolecules such as DNA, proteins and lipids, which can in turn lead to oxidative injury. Carnitine is a vitamin-like compound that serves as a carrier to transport long-chain fatty acids into the mitochondria for β-oxidation. In the present study, the effect of l-carnitine, a widely recognized essential nutrient, was evaluated on the status of lipid peroxidation and certain antioxidant enzymes and DNA damage in lymphocytes with relation to age in male wistar rats. The levels of lipid peroxides were remarkably increased whereas, the activities of antioxidant enzymes were significantly decreased in aged control animals when compared to younger controls. In aged animals, administration of l-carnitine for 21 days significantly decreased the levels of lipid peroxides and improved the activities of antioxidant enzymes like superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. l-Carnitine enhanced T-cell proliferative responses as evaluated by T-cell proliferation assay using [³H] thymidine incorporation and also significantly reduced DNA damage, apoptosis and TNF-α level in lymphocytes of aged animals. Our results suggest that l-carnitine may have a vital role in improving functions in the cells of the immune system particularly the lymphocytes possibly through its antioxidant action.     

Reversed distribution of calpains and calpastatin in human pituitary gland and selective localization of calpastatin in adrenocorticotropin-producing cells as demonstrated by immunohistochemistry

The immunohistochemical distribution of Ca2+-dependent cysteine proteinases (calpains I and II) and their endogenous inhibitor calpastatin in normal and adenomatous human pituitary tissue was studied using specific antibodies. The distributions of calpain and calpastatin were dissimilar in human pituitary gland, i.e. ACTH-immunoreactive cells were strongly positive for calpastatin and negative for calpains. PRL-, GH-, FSH-, and TSH-producing cells were negative for calpastatin, but moderately positive for calpains, especially for calpain II, the high Ca2+-requiring form of the enzyme. Similar results were found in pituitary adenoma tissue. These findings indicate that each type of cells producing a specific hormone is equipped with a different balance of the enzyme-inhibitor system involved in the Ca2+-dependent degradation of intracellular proteins.     

Production of monoclonal antibody to human esophageal cancer cell line

The immunization of Balb/C mice with esophageal cell line KYSE-50 established from poorly-differentiated esophageal squamous cell carcinoma, resulted in obtaining the monoclonal antibody KYMN-28-5. This monoclonal antibody is of the IgM class and recognizes a carbohydrate antigen contained in glycoproteins with molecular weights of 53 and 56K, and in neutral glycolipids extracted from teratomas. Tissue staining revealed that this monoclonal antibody reacts strongly with malignant tumors but only weakly, or not at all, with normal tissues, apart from squamous epithelial tissue. KYMN-28-5 is thus a useful tumor marker which will improved the accuracy of serological diagnosing squamous cell carcinoma when combined with the measurement of SCC antigen.