Hiromu Nakamura

Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus

In mammalian cells, it has been shown that adjacent multiple DNA replicons, termed a replicon cluster or a replicon domain, are replicated coordinately in a defined temporal order during the DNA synthetic (S) phase. However, no intranuclear structure of this replicon domain has been revealed in the nucleus labelled with [3H]thymidine at the limited resolution level of autoradiography. By immunofluorescent staining with antibody against 5-bromodeoxyuridine (BrdU), we succeeded in detecting novel, intranuclear ring-like structures of replicating replicon domains that were organized temporarily during the S phase of mammalian cells with incorporated BrdU.

Intracellular localization and metabolism of DNA polymerase α in human cells visualized with monoclonal antibody

Indirect immunofluorescence microscopy with monoclonal antibody against DNA polymerase alpha revealed the intranuclear localization of DNA polymerase alpha in G1, S, and G2 phases of transformed human cells, and dispersed cytoplasmic distribution during mitosis. In the quiescent, G0 phase of normal human skin fibroblasts or lymphocytes, the alpha-enzyme was barely detectable by either immunofluorescence or enzyme activity. By exposing cells to proliferation stimuli, however, DNA polymerase alpha appeared in the nuclei just prior to onset of DNA synthesis, increased rapidly during S phase, reached the maximum level at late S and G2 phases, and was then redistributed to the daughter cells through mitosis. It was also found that the increase in the amount of DNA polymerase alpha by proliferation stimuli was not affected by inhibition of DNA synthesis with aphidicolin or hydroxyurea.

Intracellular distribution of 73 000 and 72 000 dalton heat shock proteins in HeLa cells

Intracellular localization of 73,000 and 72,000 dalton heat shock proteins (HSP73/72) in HeLa cells that were heat shocked or treated with chemical stressors was investigated using indirect immunofluorescent staining. The antiserum used specifically recognized the HSP73/72 in HeLa cells, and HSPs were increased by heating cells at 42 degrees C for 2 or 4 h and by prior treatment with chemical stressors (sodium arsenite, cadmium chloride, 8-hydroxyquinoline and ethanol). There was diffuse cytoplasmic staining at 37 degrees C, whereas nucleoli were stained brightly when cells were heated at 42 degrees C for 2 h. This rapid accumulation of HSP73/72 in the nucleoli was not inhibited by cycloheximide (50 micrograms/ml). Translocation of HSPs to the nucleoli was specific for heat because no translocation was induced by treatment with chemical stressors. When the cells were returned to 37 degrees C after heating, the HSPs in their nucleoli disappeared rapidly and diffuse cytoplasmic staining was present after 6-9 h. Our results suggest that the transient accumulation of HSP73/72 in HeLa cell nucleoli that is induced by heat shock is not correlated with the development of thermotolerance obtained in other cell systems.

Possible cytoskeletal association of 69,000- and 68,000-dalton heat shock proteins and structural relations among heat shock proteins in murine mastocytoma cells.

When murine mastocytoma cells (FMA 1) were heat shocked (42 degrees C for 4 h), nine heat shock proteins (HSPs) were detected by two-dimensional gel electrophoresis. Their apparent molecular weights were 100, 85, 69, 68, 32, 30, and 23 kDa (3 of 23 kDa). The structural homology of 4, 69, 68, 32, and 30 kDa, was demonstrated by two-dimensional tryptic peptide mapping. The 69- and 68-kDa HSPs were purified and rabbit antisera against these HSPs were prepared. A small fraction (less than 10%) of the 69- and 68-kDa HSPs were copurified with the microtubules and were present in the Triton X-100/KCl cytoskeletal fraction as shown by immunoblotting with the antiserum and by peptide mapping. Our results are consistent with the hypothesis of a cytoskeletal role for HSPs.

Localization of 350K molecular weight and related proteins in both the cytoskeleton and nuclear flecks that increase during G1 phase.

Monoclonal and polyclonal antibodies were raised against the highest molecular weight microtubule-associated protein (MAP-1) isolated from brain. Immunoblotting with the antibodies revealed the presence of cross-reactive protein of 350K or less on whole cells, isolated nuclei and cellular microtubules. Two-dimensional peptide maps showed substantial homology of immunoprecipitated cellular proteins of 350K, 80K and 51K with a 25K fragment of brain MAP-1. On antibody staining, immunofluorescence was seen on a cytoplasmic network, the mitotic spindle, the centrosome, and intranuclear flecks. The antibody causing immunofluorescence in all these sites was absorbed most effectively with slices of blotted membrane which contained the 350K protein. These results suggest that the cross-reactive molecules in diverse sites belong to the family of the 350K protein. The number of nuclear flecks and the amount of bound radioactivity of 125I-antibody almost doubled during G1 phase.

Nuclear immunofluorescence by a monoclonal antibody against microtubule-associated protein-1 as it is associated with cell proliferation and transformation

Monoclonal antibody against microtubule-associated protein-1 produced intranuclear immunofluorescent spots, which disappeared under growth-inhibited conditions caused by serum starvation and saturated cell density in untransformed cells. A change of medium to 10% serum gave rise to the reappearance of nuclear spots before the resumption of DNA synthesis. This reversible change of immunofluorescence was also caused by a temperature shift in rat 3Y1 cells transformed by Simian virus-40-A640 (temperature-sensitive in large T-antigen). The fluorescence decreased during S phase of the cell cycle. In contrast the transformed cells always showed nuclear fluorescence, irrespective of serum concentrations or the cell cycle. Growth-inhibited cells previously treated with detergent and salt revealed nuclear fluorescent spots. This result suggested antigenic modification.

Rapid phosphorylation of MAP-2-related cytoplasmic and nuclear Mr 300,000 protein by serine kinases after growth stimulation in quiescent cells☆

Antibody against brain microtubule-associated protein 2 (MAP-2) immunoprecipitated Mr 300,000 and 80,000 proteins of cultured fibroblasts and kidney cells. These proteins were not appreciably phosphorylated in quiescent cells, but were rapidly phosphorylated after growth stimulation by insulin, epidermal and fibroblast growth factors, transferrin, phorbol ester and diacylglycerol in the presence of Ca2+, in a manner similar to that of MAP-1-related Mr 350,000 protein (J. Cell Biol. 100, 748-753). A Ca2+ ionophore, which is known to make the quiescent cell competent but not to enter into the growth cycle, did not induce the phosphorylation. In a chase experiment, decay half lives of labeled phosphoproteins were 5 h for Mr 350,000 and 300,000 proteins, and 1.5 h for Mr 80,000 protein. On subcellular fractionation, phosphorylated Mr 350,000 and 300,000 proteins were detected first mainly in the cytoplasm and then in the nucleus, while Mr 80,000 phosphoprotein was consistently detected in the cytoplasm. The phosphorylation of these proteins occurred on serine residues after stimulation with various factors. Thus, the phosphorylation of cytoskeleton-associated Mr 350,000 and 300,000 proteins by serine kinases seems to be a common second process after growth stimulation and to link cytoplasmic and intranuclear events.

Phosphorylated 350 kD protein in the nucleus as it is associated with cell transformation

Protein kinases are thought to play a key role in signal transduction and oncogenesis, but little is known about the intranuclear phosphorylation events associated with transformation. Here we report on cell cycle-dependent phosphorylation of cytoskeleton-associated 350 kD protein and the regular interchange in its location between the nucleus and cytoplasm of normal cells. Persistent intranuclear location of the phosphorylated 350 kD protein was also found throughout the cell cycle in transformed cells, as detected by immunoprecipitation of 32P-phosphorylated 350 kD protein from isolated nuclei and immunofluorescent staining with a monoclonal antibody that recognized phosphorylated site of 350 kD protein. A conditional transformed phenotype induced by a temperature-sensitive (ts) viral oncogene or a transforming growth factor was also associated with the intranuclear presence of the phosphorylated 350 kD protein. Thus the 350 kD protein seems to be a target molecule of protein kinases that are stimulated directly or indirectly by growth factors or by oncogene products in the nucleus, and appears to be a new transformation-related nuclear antigen.

Growth of a cultured leukemia subline was promoted by conditioned medium of thymic reticuloepithelial-like cells (B6TE)☆

A murine leukemia subline (L17R) was selectively developed in the presence of conditioned medium of a thymic reticuloepithelial-like cell line (B6TE). Cytotoxicity tests and immunofluorescence microscopy showed that L17R cells were negative in the expression of Thy 1.1, Lyt 1.2 and terminal deoxynucleotidyl transferase (TdT), however, 35% positive in Lyt 2.1 phenotype, and 95% positive in the expression of peanut agglutinin (PNA) receptor. B6TE conditioned medium had no activity of interleukin 1 (IL 1), interleukin 2 (IL 2), interleukin 3 (IL 3) and granulocyte/macrophage colony-stimulating factor (GM-CSF). When L17R leukemic cells were plated at a low cell density, their growth was accelerated 40 times by the addition of concentrated B6TE culture supernatant. This growth activity, tentatively designated leukemia-growth-promoting factor (LGPF), was heat sensitive, and its mol. wt was estimated to be approx. 25,000 from the elution pattern of Sephadex G-100 chromatography.

Isolation and characteristics of a leukemia-growth-promoting factor from calf thymus.

Leukemia-growth-promoting factor (LGPF) as we previously reported stimulates the growth of a murine leukemia subline (L17R) extensively. LGPF was isolated and 10(4) fold purified from calf thymuses by a combination of ammonium sulfate precipitation, Sephadex G-100 chromatography, hydroxylapatite chromatography, and Mono S-fast protein liquid chromatography (Mono S-FPLC). The mol. wt of LGPF was estimated to be approximately 25,000 a.m.u. from the elution pattern of Sephadex G-100 chromatography. The activity had high affinity for Mono S beads which are cation exchangers. Mono S fractions of LGPF are effective at a low concentration of 5 ng/ml. The activity was inactivated by heat (56 degrees C, 30 min), 1 mg/ml trypsin (37 degrees C, 1h), and 50 mM dithiothreitol (20 degrees C, 1h). The growth L17R leukemia cells are not only stimulated by LGPF, but also by pituitary and brain fibroblast growth factor (FGF). These data strongly suggest that LGPF is a heat sensitive cationic protein(s) acting as a member of FGF family.