The use of A23187 as a biochemical tool is described. A23187 was used to elucidate the mechanisms of intracellular transport and secretion of N-hydroxylated aniline metabolites in rat hepatocyte primary culture. Results indicate a membrane-bound intracellular transport. Also, A23187 induced macrophages in their anti-tumour cytostatic activity using P815 tumour cells in in vitro co-cultures of macrophages and tumour cells. Results indicate the activating role of A23187 in macrophage leukotriene ?4 release and enhanced macrophage anti-tumour activity. In conclusion, A23187 proved to be a useful tool in studying calcium dependent metabolic processes.
A clonal derivative named CL6 has been isolated from pluripotent P19 embryonal carcinoma (EC) cells, after long term culture under conditions for mesodermal differentiation. The CL6 subline has a morphology similar to P19 cells and grows exponentially in standard medium, but unlike P19 cells, it efficiently differentiates into beating cardiac muscle in adherent culture with 1% dimethyl sulfoxide (DMSO). During differentiation, CL6 cells displayed the following general cardiac muscle properties: (1) α- and β-cardiac myosin heavy chain (MHC) transcripts were first detected on day 10 after treatment with DMSO. (2) A sarcomere MHC protein also appeared on day 10, simultaneously with the initiation of contraction. (3) Immunofluorescence analysis showed that vigorously contracting cells have a striated structure. (4) Skeletal muscle specific transcripts, such as myogenin and MyoD, were not detected throughout differentiation. On the other hand, in suspension culture with 1 μM retinoic acid (RA), the condition for neural differentiation of P19 cells, the CL6 cells developed into neurons with poor outgrowth. Taken together, the CL6 subline seems not to be committed to a mesodermal lineage but to represent a developmental stage closer to differentiated cardiac muscle than the P19 cell line. Since CL6 cells directly differentiate into cardiac muscle in adherent culture, it is the most useful cell line for studying the differentiation of cardiac muscle in vitro.
Many studies on in vitro transformation of human cells indicate that the cells must be immortalized before they can be neoplastically transformed, indicating that immortalization is a critical step in multistep neoplastic transformation of human cells. We immortalized three human cell lines by repeated treatment with either 60Co gamma rays or a chemical carcinogen, 4-nitroquinoline 1-oxide, and found that all three immortalized cell lines have mutations in the tumor suppressor gene, p53. Direct sequencing of the reverse-transcribed mRNA and immunoprecipitation of p53 protein revealed that mutant p53 is selectively expressed in all the immortalized cell lines, whereas the genomic fragments of the immortalized cells contain wild-type and mutated p53 alleles. Although the mutated p53 is selectively expressed in the immortalized cells, expression of the wild-type p53 was induced by treatment of the cells with a hypomethylating reagent, 5-azacytidine, indicating that the wild-type p53 allele might be inactivated by hypermethylation of DNA. Actually, the entire genomic locus including the promoter region of p53 is hypermethylated in all the immortalized cell lines. Expression and phosphorylation of Rb was normal in these three cell lines. Thus, inactivation of both wild type p53 alleles and selective expression of mutated p53 seem to be key factors in the immortalization of human fibroblasts.
We reported that immunoelectron microscopy was an excellent tool for determining the subcellular localization of thiolase isozymes, acetoacetyl-CoA thiolase (T-I) and 3-ketoacyl-CoA thiolase (T-III) in n-alkane-grown Candida tropicalis cells (KAMASAWA, N. et al., (1992). Cell Struct. Funct., 17: 203-207). Current investigation on the visualization of other peroxisomal enzymes, acyl-CoA oxidase (ACO), catalase (KAT), carnitine acetyltransferase (CAT), isocitrate lyase (ICL) and malate synthase (MS), showed that ACO localized in peroxisomes, KAT in peroxisomes and cytoplasm, and CAT in peroxisomes, mitochondria and cytoplasm. Most of ICL and MS were found in peroxisomes. These results agreed with previous biochemical studies and supported the presumed roles of these enzymes. The same technique was applied to study the process of synthesis and localization of these enzymes early in the cultivation period in n-alkane medium when peroxisomes began to proliferate. ACO and T-III were rapidly induced after transfer of cells from glucose- to n-alkanemedia. There was a drastic change of their location from cytoplasm to peroxisomes between 1 h and 2 h after the transfer, while T-I, KAT and CAT were moderately induced in cytoplasm and their location was gradually changed to each organelle. ICL and MS, the key enzymes in the glyoxylate cycle, were already localized in peroxisomes in the glucose-grown cells and respective inducible enzymes also were gradually localized there. This visual analysis is useful for the vivid elucidation of the process of peroxisome proliferation and enzyme transport within a cell.
We showed that DNA-dependent ATPase Ql (DNA helicase Ql) from xeroderma pignientosiim complementation group C (XP-C) cells elutes from FPLC Mono Q column at higher concentrations of KC1 than that from other human cells (35). We purified DNA helicase Ql from XP-C and HeLa cells. The purified fractions of both cells contained a major polypeptide with a molecular mass of 73 kDa and had the same enzymatic properties, including salt- and temperature-sensitivity. Characterization using an anti-DNA helicase Ql antibody indicated that this enzyme localized in the nuclei and was not modified by incorporating phosphate groups through phosphorylation and ADP-ribosylation. No interactions of DNA helicase Ql with other proteins were indicated by immunoprecipitation of the helicase from crude extracts. No difference was observed in XP-C cells in intracellular localization of DNA helicase Ql, phosphorylation, and the interaction with other proteins as compared to HeLa cells.
Orthogonal aggregates of small intramembrane particles, termed "assemblies, " are concentrated especially in the cell membranes of astrocytic processes that form the glia limitans at the outer surface of the brain and the perivascular sheath surrounding the parenchymal blood vessels. As an initial step to clarifying the totally unknown biochemical nature of this intramembrane structure, we have devised a culture system which enhances the differentiation of assemblies in secondary cultures of astrocytes derived from neonatal mouse neopallium. Since assemblies are most concentrated in the plasma membranes attaching to the basement membrane, we expected that extracellular matrix molecules constituting the basement membrane would be suitable candidates for our aim. We report here that a mixture of type IV collagen, laminin, and fibronectin, major components of the extracellular matrix, has the potency to increase assembly density in cultured astrocytes. We also report that, in freeze-fracture electron microscopy of cultured cells, one can satisfactorily preserve membrane structure and reliably obtain large replicas by inoculating cells on aluminium foil and peeling it from the cells in a freeze-fracture apparatus.
NCR-G3 cells were established from a testicular embryonal carcinoma and were differentiated into multi-lineages including trophectoderm cells by exposure to retinoic acid. The differentiated cells began to produce human chorionic gonadotropin (hCG), a trophectoderm-specific hormone, which was regulated at the mRNA level. As we assumed that genes responsible for differentiation were differentially expressed at the early stage of retinoic acid-induced differentiation, we prepared a cDNA library from retinoic acid-treated NCR-G3 cells. This cDNA library was then screened for genes whose expression was induced during the differentiation of these cells. From about 5 × 104 clones screened, three independent sequences were isolated. Sequencing analysis revealed that clone 1002 codes for mcll/EAT, which has a Bcl-2 homology domain. The expression of mcll/EAT, the Bcl-2 related gene, was increased at an early stage of the retinoic acid-induced differentiation and preceded the up-regulation of cytokeratin and hCG genes after ratinoic acid treatment. Furthermore, mcll/EAT was also up-regulated by heat shock, which has recently been shown to induce the cells to differentiate.
Effects of nitric oxide (NO) on oxygen uptake of Ehrlich ascites tumor cells (EATC) were examined in a study of the biological actions of NO on respiration and energy metabolism at the cellular level. Endogenous respiration of EATC was inhibited reversibly by NO in a dose dependent manner. Oxyhemoglobin, an NO trapping agent, restored the respiration promptly. The inhibitory action of NO also depended on oxygenconcentration, and the duration of suppression was prolonged remarkably at low oxygen tension. Similar inhibition was also observed in the presence of glucose. In this case, both lactate production and glucose consumption were promoted by NOC 18, an NO generating agent, and the activation was enhanced by lowering the oxygenconcentration. Furthermore, the membrane potential of EATC was depolarized transiently by adding NO, and the degree of depolarization was decreased in the presence of glucose. These results suggest that at physiologically low oxygen tension in ascites fluid, NO acts not only as a cytotoxic respiratory inhibitor but also as a regulatory factor in the energy metabolism of EATC.