[Cultured Cells in the Aging Research, Exhibiting Cell Surface Component Functions, Intracellular Signaling, a Novel Adaptor Molecule, Aging Phenotype Expression and Various Aspects of the Cellular Physiology].

Cellular aging is one of the most extraordinary phenomena that mammalian cells undergo in vivo and in vitro. We have been observing their behavior for approximately 4 decades and here would like to summarize some of our salient findings. Normal cells such as human diploid cells exhibit finite growth potential in vitro as well as a set of senescent cell phenotypes.

TNF-alpha from monocyte of patients with pre-eclampsia-induced apoptosis in human trophoblast cell line.

Objective: In pre-eclampsia, fetal growth restriction is frequently observed, and the possible involvement of inhibitory substances on trophoblast cell proliferation and differentiation has been suggested. The objective of this study was to investigate the effects humoral factors, such as cytokines, produced in immune cells on proliferation of an immortalized trophoblastic cell line (TCL) that we established.

Methods: Serum and lymphocyte layers were isolated from the blood of normal pregnant and preeclamptic women.

Thyme (Thymus vulgaris L.) leaves and its constituents increase the activities of xenobiotic-metabolizing enzymes in mouse liver.

The effects of thyme (Thymus vulgaris L.) leaves and its phenolic compounds, thymol and carvacrol, on the activities of xenobiotic-metabolizing enzymes, i.e.

A positive role of phosphatidylinositol 3-kinase in aging phenotype expression in cultured human diploid fibroblasts.

In order to detect the role that phosphatidylinositol 3-kinase (PI3K) plays in the aging of human diploid fibroblasts, we analyzed cellular inositol phospholipids and expression of PI3Ks. In aged cells a decrease in phosphatidylinositol 3,4-bisphosphate (PI3,4P(2)) was notable, while phosphatidylinositol 3-phosphate (PI3P) and phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)) decreased slightly. On the other hand, the messages of PI3K IIalpha, Vps34, and p110delta decreased and that of PI3K IIbeta increased during aging.

Cytoskeletal reorganization induced by insulin: involvement of Grb2/Ash, Ras and phosphatidylinositol 3-kinase signalling.

Background: Cytoskeletal reorganization is important for a wide variety of insulin-mediated biological actions, including cell growth, migration and metabolism, but the intracellular signalling pathways leading to insulin-induced cytoskeletal reorganization have largely been unknown. We therefore investigated the involvement of Grb2/Ash-Ras and phosphatidylinositol (PI) 3-kinase in the insulin-induced morphological changes in fibroblasts over-expressing human insulin receptors (HIRcB cells).

Results: Insulin, as well as 12-O-tetradecanoylphorbol-13-acetate (TPA) and 8-bromo-cAMP, induced a unique morphological change associated with actin cytoskeletal reorganization characterized by the disruption of actin stress fibres and thicker actin bundle formation.

Telomerase positive human diploid fibroblasts are resistant to replicative senescence but not premature senescence induced by chemical reagents.

Human diploid fibroblasts in tissue culture undergo replicative senescence after a finite number of divisions that is characterized by a permanent loss of their dividing potential. However, senescence-like phenotypes, including growth cessation, morphological changes, and appearance of senescence-associated beta-galactosidae (SA-gal) activity, can be induced by treating early passage cells with C(6)-ceramide, H(2)O(2), LY294002, or trichostatin A. While there is convincing evidence that telomere shortening is causally related to replicative senescence, the role of telomere shortening in the chemical-induced premature senescence is unclear.

Transcriptional regulation of cellular ageing by the CCAAT box-binding factor CBF/NF-Y.

Cellular ageing is a systematic process affecting the entirety of cell structure and function. Since changes in gene expression are extensive and global during ageing, involvement of general transcription regulators in the phenomenon is likely. Here, we focus on NF-Y, the major CCAAT box-binding factor, which exerts differential regulation on a wide variety of genes through its interaction with the CCAAT box present in as many as 25% of the eukaryotic genes.

Rapid reversion of aging phenotypes by nicotinamide through possible modulation of histone acetylation.

Aging appears to be an irreversible process. Here we report that nicotinamide (NAA) can induce rapid and reversible reversion of aging phenotypes in human diploid fibroblasts in terms of cell morphology and senescence-associated beta-galactosidase activity. Although NAA seems to enhance the replicative potential of the cells, it has little effect on their growth rate and life span, suggesting that NAA action is rather separated from the cellular replicative system.

Possible role of subunit A of nuclear factor Y (NF-YA) in normal human diploid fibroblasts during senescence.

NF-Y, a heterotrimeric CCAAT binding protein, may have a role in regulating some G1/S genes whose expressions are attenuated during replicative senescence [Matuoka and Chen (1999) Exp Cell Res 253: 365-371]. The hallmark of replicative senescence is the loss of dividing potential. Hence, attenuation of G1/S gene expressions may be causally related to aging.

Expression of the SART3 tumor rejection antigen in renal cell carcinoma.

Purpose: We recently reported that SART3 tumor rejection antigen is recognized by HLA class I restricted cytotoxic T lymphocytes from patients with esophageal cancer. We now investigate the expression of SART3 antigen in renal cell carcinoma to identify an appropriate molecule that may be used in specific immunotherapy of renal cell carcinoma.

Materials And Methods: Renal cell carcinoma and nontumorous kidney tissues were obtained at surgery.

Nuclear factor Y (NF-Y) and cellular senescence.

NF-Y, also termed CBF, is a major CCAAT-binding transcription factor that specifically recognizes the consensus sequence 5'-CTGATTGGYYRR-3 or 5'-YYRRCCAATCAG-3' (Y = pyrimidines and R = purines) present in the promoter region of many constitutive, inducible, and cell-cycle-dependent eukaryotic genes. The functional NF-Y is a heterotrimeric protein, consisting of three different subunits, A, B, and C. Each of the three subunits contains two or three distinct protein-interacting domains for trimer formation and for interacting with other nuclear proteins.

Downregulated expression of the signaling molecules Nck, c-Crk, Grb2/Ash, PI 3-kinase p110 alpha and WRN during fibroblast aging in vitro.

An RT-PCR analysis was performed to examine changes in intracellular signal transducing molecules during in-vitro aging of human fibroblasts. Expression of Nck, c-Crk, Grb2/Ash, phosphoinositide (PI) 3-kinase p110 alpha and Werner's syndrome gene product WRN was noticeably reduced in late passage cells, showing a concurrent downregulation of a set of signaling molecules accompanying aging.

A novel ligand for an SH3 domain of the adaptor protein Nck bears an SH2 domain and nuclear signaling motifs.

Nck is a small protein composed of Src homology regions (SH) 2 and 3, paralleling the adaptors c-Crk and Grb2/Ash, but its function remains enigmatic. To clarify Nck signaling, a human brain cDNA library was searched for targets of the SH3 moiety of Nck. A novel molecule detected therefrom (referred to as Nck-, Ash- and phospholipase Cgamma-binding protein 4) contained proline-rich sequences and, through the function of one of them, interacted with the middle SH3 domain of Nck.

Interaction between the amino-terminal SH3 domain of CRK and its natural target proteins.

CRK is a human homolog of chichen v-Crk, which is an adaptor protein. The SH2 domain of CRK binds to several tyrosine-phosphorylated proteins, including the epidermal growth factor receptor, p130(Cas), Shc, and paxillin. The SH3 domain, in turn, binds to cytosolic proteins of 135-145, 160, 180, and 220 kDa.

Splicing isoforms of rat Ash/Grb2. Isolation and characterization of the cDNA and genomic DNA clones and implications for the physiological roles of the isoforms.

We obtained three types of cDNA clones homologous to Ash/Grb2(Ash-l) cDNA from rats. One of these clones, Ash-psi, was an unusual transcribed gene having 93% identity in the nucleotide sequence to Ash-l. The other two clones, Ash-m and -s, had nucleotide sequences identical with Ash-l cDNA in the amino-terminal region.

Grb2/Ash binds directly to tyrosines 1068 and 1086 and indirectly to tyrosine 1148 of activated human epidermal growth factor receptors in intact cells.

The activation of receptor tyrosine kinases generates tyrosine-phosphorylated recognition motifs for the binding of signaling proteins containing Src homology 2 domains. We determined the binding sites of Grb2/Ash, an Src homology 2 domain-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing human EGF receptor mutants in which one of the autophosphorylation sites was retained. In intact cells, the amount of Grb2/Ash coimmunoprecipitated with mutant receptors retaining tyrosines 992, 1068, 1086, 1148, or 1173 was approximately 10, 85, 55, 50, or 20% of wild-type levels, respectively.

C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins.

CRK protein, together with GRB2/ASH and Nck proteins, belongs to the adaptor-type Src homology (SH)2-containing molecules, which transduce signals from tyrosine kinases. Here another guanine nucleotide-releasing protein (GNRP), C3G, has been identified as a CRK SH3-binding protein. The nucleotide sequence of a 4.

Different interactions of Grb2/Ash molecule with the NGF and EGF receptors in rat pheochromocytoma PC12 cells.

We have previously shown that nerve growth factor (NGF) induces a rapid and relatively continuous activation of Ras in rat pheochromocytoma PC12 cells while epidermal growth factor (EGF) activates Ras transiently, and that tyrosine kinase activity of the NGF receptor is essential for the activation of Ras (Muroya et al., Oncogene, 7, 277-281, 1992). In order to explore the signaling mechanism from tyrosine kinase to Ras activation in more detail, interactions between two adaptor molecules, Shc and Grb2/Ash, which contain Src homology regions, and their interactions with the NGF and EGF receptors were examined.

Association of Ash/Grb-2 with dynamin through the Src homology 3 domain.

Ash/Grb-2 is an adaptor protein composed only of Src homology (SH) 2 and SH3 domains that is considered to be essential for Ras activation. To clarify the downstream of Ash signaling, we investigated Ash-bound proteins. Ash-glutathione S-transferase (GST) fusion proteins were used to affinity-purify proteins bound to Ash.

Growth factors differentially stimulate the phosphorylation of Shc proteins and their association with Grb2 in PC-12 pheochromocytoma cells.

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including two proteins, Shc and Grb2, that are thought to lie upstream from the ras protooncogene in a signaling cascade. The phosphorylation and molecular associations of these proteins were evaluated in PC-12 pheochromocytoma cells treated with nerve growth factor (NGF), epidermal growth factor (EGF), and insulin. Both NGF and EGF stimulated the tyrosine phosphorylation of Shc proteins and their subsequent association with the receptors.

Ash/Grb-2, a SH2/SH3-containing protein, couples to signaling for mitogenesis and cytoskeletal reorganization by EGF and PDGF.

The Src homology (SH) region 2 binds to phosphorylated tyrosine residues and SH3 domains may interact with cytoskeletal molecules and GTPase-activating proteins for Rho/Rac proteins (the small GTP-binding proteins related to Ras). The recently cloned Ash/Grb-2 protein, a 25-28 kDa molecule composed entirely of SH2 and SH3 domains, is a mammalian homolog of the Caenorhabditis elegans Sem-5 protein, which communicates between a receptor protein tyrosine kinase and a Ras protein. In the present study the function of Ash/Grb-2 was investigated by microinjecting cells with an anti-Ash antibody.

Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2.

Insulin activates the ras proto-oncogene product p21ras (Ras) by stimulating conversion of the inactive GDP-bound form of Ras to the active GTP-bound form. The protein ASH (for abundant Src homology) (Matuoka, K., Shibata, M.

Diethylnitrosamine- and partial hepatectomy-induced decrease in alpha 2u-globulin mRNA level in the rat liver.

Changes in gene expression in the rat liver were investigated by analyzing cDNA libraries for liver mRNAs from adult male rats injected with a chemical carcinogen diethylnitrosamine (DEN). Differential screening using normal and DEN-treated liver cDNAs as probes demonstrated that some of the mRNA species had noticeably lower abundance in the DEN-treated liver than in the untreated liver. Surprisingly, most of those clones were found to code for alpha 2u-globulin (A2uG), an abundant protein in the male rat liver.