Hyaluronate synthetase inhibition by normal and transformed human fibroblasts during growth reduction.

To establish the relation of glycosaminoglycan synthesis to cell proliferation, we investigated the synthesis of individual glycosaminoglycan species by intact cells and in a cell-free system, using normal and transformed human fibroblasts under differing culture conditions. Reducing serum concentration brought about a marked decline in the synthesis of hyaluronate (HA) as well as cell proliferation on both normal and transformed cells. Both HA synthesis and proliferation decreased with increasing cell densities markedly (in inverse proportion to cell density) in normal cells but gradually in transformed cells. This noticeable congruity of the changes in HA synthesis and proliferation indicates that the change in HA synthesis is related primarily to cell proliferation rather than to cell density or cellular transformation. Examination of HA synthesis in a cell-free system demonstrated that the activity of HA synthetase also fluctuated in conjunction with cell proliferation. Furthermore, growth-reduced cells (except crowded transformed cells) inhibited cell-free HA synthesis and this inhibition was induced coincidentally with a decrease in both HA synthetase activity and proliferation. These findings suggest that the change in HA synthesis is significant in the regulation of cell proliferation.

Mitogenesis in response to PDGF and bombesin abolished by microinjection of antibody to PIP2.

The turnover of phosphatidylinositol 4,5-bisphosphate (PIP2) is believed to constitute a crucial step in the signaling pathways for stimulation of cells by a variety of bioactive substances, including mitogens, but decisive evidence for the idea has not been obtained. In the present study, a monoclonal antibody to PIP2 was microinjected into the cytoplasm of NIH 3T3 cells before or after exposure to mitogens. The antibody completely abolished nuclear labeling with [3H]thymidine induced by platelet-derived growth factor and bombesin, but not by fibroblast growth factor, epidermal growth factor, insulin, or serum. The findings strongly suggest that PIP2 breakdown is crucial in the elicitation and sustaining of cell proliferation induced by some types of mitogens such as platelet-derived growth factor and bombesin.

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. Both NGF and EGF induced rapid tyrosine phosphorylation of Shc and its association with both the receptors and with Grb2/Ash. When cells were stimulated with EGF at 4 degrees C, the activation of Ras proceeded slowly and MAP kinase activation was quite low. Under such restricted conditions, tyrosine-phosphorylated Shc formed a complex with Grb2/Ash, suggesting that the complex formation may be one of the immediate early responses. In contrast to Shc, Grb2/Ash bound to EGF receptor but did not form a stable complex with the NGF receptor. These results suggest that there may be an alternative pathway for the activation of Ras in PC12 cells.

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.

Changes in cell-surface glycosaminoglycans in human diploid fibroblasts during in vitro aging.

Changes in glycosaminoglycans during in vitro aging were investigated in human diploid fibroblasts. The cells were found to produce predominantly hyaluronate and smaller amounts of chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and heparan sulfate. Accumulation of heparan sulfate on the cell surface was notable during aging. Total glycosaminoglycan production in preconfluent culture did not change with population doubling level (PDL), while in confluent culture a decline in glycosaminoglycan production was observed. In contrast with this, heparan sulfate on the cell surface increased as a function of PDL in both confluent and preconfluent cultures. The distribution pattern of heparan sulfate in medium and cell surface indicated that the increase in heparan sulfate on the cell surface could be attributed to an increased accumulation on the cell surface, but not to an elevated production. Thus, we conclude that the increased accumulation of heparan sulfate on the cell surface might be involved in an age-related alteration in the cell membrane.

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. Further analysis by in situ hybridization revealed that the decrease in the A2uG mRNA level occurred in the area where liver cells were proliferating due to DEN treatment and/or partial hepatectomy (PH). The findings indicate coincidence of cell proliferation with a decrease in the A2uG gene expression in the adult male rat liver, implying that the A2uG-related change favors chemical carcinogen-induced cell growth.

Cell surface changes in senescent and Werner's syndrome fibroblasts: their role in cell proliferation.

Cell surface is known to participate in the regulation of cell proliferation through interaction with adjacent cell surfaces or the extracellular matrix, or both. A clinical survey of the Werner syndrome suggests some disorders in glycosaminoglycan metabolism. Also, the skin fibroblasts derived from the patients with WS have a reduced proliferation capacity. We here examined, in vitro and in vivo, alterations of the cell-surface properties of WS cells and aging human fibroblasts. Cell-surface negative charges, examined by electrophoretic mobility of dispersed single cells in buffer, were seen to decline steadily as a function of cumulative population doublings. A strict linear relationship was found between electrophoretic mobility (micron/sec/V/cm) and number of cells harvested at each passage in all cell lines examined. The slope of this line in cells from donors of different ages indicated that WS fibroblasts resemble cells from much older normal controls. The same conclusion was drawn from our previous study of Con A-mediated red cell adsorption, which was confirmed as reflecting an alteration of cell-surface coat negative charge. Electrophoretic mobility after treatment of cell surface with degradative enzymes showed that the cell-surface negative charges were attributable to sialic acid, chondroitin sulphates, hyaluronic acid, and heparan sulphate. Two-dimensional electrophoresis of 3H-glucosamine incorporated glycosaminoglycans (GAGs) revealed that heparan sulphate was the main component of GAGs on the fibroblast cell surface and that the relative amount of heparan sulphate among GAGs on the cell surface increased in vitro with the number of passages. Growth kinetics of fibroblasts on sheets of fixed cells treated with a fixative (glutaraldehyde) and degradative enzymes were examined to elucidate the role of cell-surface GAGs in the regulation of cell proliferation. Cell growth was inhibited 40% when the fibroblasts were cultured on the fixed sheets of late passage cells. Treatment of the fixed cell sheets with heparitinase or nitrous acid resulted in complete recovery from the growth inhibition. Cell growth on sheets of fixed cells derived from young, middle, and senescent fibroblasts showed that the surface of the senescent cells had the greatest inhibitory effect. These inhibitory effects of fixed cell sheets correlated well with both the amount of heparan sulphate relative to the total GAGs on the surface and to the saturation density of cell growth at each passage. These findings strongly suggest that heparan sulphate, or its complex, on the cell surface is involved in the regulation of cell proliferation.(ABSTRACT TRUNCATED AT 400 WORDS)

[Milk of calcium renal stone: a case report].

A case of milk of calcium renal stone is presented. A 29-year-old woman complained of fever up and left lateral abdominal pain was diagnosis of milk of calcium renal stone. Ultrasonography showed hemispheric hypoechoic area and horizontal high echoic line with acoustic shadow. So-called "milk of calcium renal stone" is relatively uncommon and reports of ultrasonographic features are rare.

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. To understand how NF-Y is involved in regulating G1/S genes during replicative senescence, we have examined the expressions of three NF-Y subunit genes in human IMR-90 cells over the entire course of their life-span. The mRNA levels of NF-YA, B, and C did not show any age-dependent change. In contrast, the protein level of NF-YA exhibited a significant and progressive decrease during cell senescence. Cross-linking experiments indicated that NF-Y may interact with proteins such as GCN5 and P/CAF. Co-transfection of cells with plasmid encoding NF-YA protein enhanced the expression of reporter gene fused with G1/S gene promoter that contains NF-Y sites. In contrast, co-transfection with plasmid encoding the dominant negative NF-YA mutant suppressed the expression of the reporter genes. Transient transfection of human cells with dominant negative NF-YA mutant could lead to an increase in neutral beta-galactosidase activity, a marker of cell senescence. These results support the view that NF-Y may have a role in cell senescence.

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. Only the trimeric NF-Y, and not the individual subunit, possess DNA-binding activity. The transcriptional activity of NF-Y can be regulated by differential expression, alternative splicing, protein-protein interactions, and cellular redox potential. The regulation of thymidine kinase (TK) and dihydrofolate reductase (DHFR) genes in human diploid fibroblasts serves as an example of how NF-Y may have a role in replicative senescence by regulating age-dependent G1/S genes.

Heparan sulfate enhances growth of transformed human cells.

Previous studies showed that cell surface heparan sulfate (HS) is involved in density-dependent growth regulation of normal human cells. In this study the effects of HS on proliferation of transformed cells were studied in vitro. Exogenously added HS prepared from normal tissues (rat kidney and bovine kidney) enhanced growth of transformed human and animal cells (gamma ray- or virus-transformed WI-38, and HeLa cells and chemically induced mouse hepatoma cells), but inhibited that of normal human and animal cells (WI-38, 3T3, and rabbit liver cells). HS was less effective on growth of both normal and transformed human cells at higher cell density. Although the exogenous HS did not bind to cells tightly, HS was found to affect cell growth not by modulation of growth-related substances in the medium, but through contact with the cell surface. HS preparation from tumour cells (mouse hepatoma cells) exerted similar effects on cell growth. Heparin, structurally similar to HS, inhibited growth of both normal and transformed human cells. These findings suggest that: 1. HS plays a particular function in contact regulation of cell proliferation. 2. Transformation-related changes in the structure of HS molecules do not much affect the function of HS. 3. The cellular transformation, however, is accompanied by alteration in the growth-regulating system sensitive to extracellular HS.

Growth-coupled changes in glucosaminoglycans (heparan sulfate and hyaluronic acid) in normal and transformed human fibroblasts.

Changes in glycosaminoglycans (GAGs) were investigated in relation to cell density, growth and transformation of human fibroblasts. Relative amounts (percentages of the total GAGs) of heparan sulfate (HS) increased and those of hyaluronic acid (HA) decreased in growth-reduced (serum-starved, exogenous HS-treated and dense) cultures of normal (WI-38) cells. In contrast, transformed (WI-38 CT-1) cells exerted such GAG changes only in serum-starved cultures, but not in HS-treated or dense cultures. These results indicate that the changes in glucosaminoglycans (G1cAGs) (HS and HA) is coupled exclusively with cell growth.

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. The antibody abolished both S phase entry and the reorganization of actin assembly to ruffle formation upon stimulation with epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). On the other hand, anti-Ash antibody had no effect on S phase entry or actin stress fiber formation induced by either serum or lysophosphatidic acid. Since the induction of DNA synthesis, ruffle induction and stress fiber formation involve a function of Ras, Rac activation and Rho activation respectively, the findings strongly suggest that Ash plays a critical role in the signaling of both pathways downstream from growth factor receptors to Ras and Rac. Consistent with this, Ash co-precipitated with EGF receptor from EGF-stimulated cells. Other proteins of approximately 21, 29, 135 and 160 kDa were also detected in the anti-Ash antibody immunoprecipitates, suggesting a role of Ash as a linker molecule in signal transduction downstream of growth factor receptors.

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. In contrast, insulin had no effect on Shc phosphorylation, despite the expression of functional insulin receptors in these cells at levels comparable to those observed for NGF and EGF. NGF and EGF also induced the association of Shc proteins with a Grb2 fusion protein or endogenous Grb2, whereas insulin had no effect. All of the tyrosine-phosphorylated Shc proteins associated with the Grb2 fusion protein, although only about half of the endogenous Shc was phosphorylated in response to NGF or EGF. However, all three hormones induced the association of several additional tyrosine phosphorylated proteins with Grb2, some of which also coprecipitated with antiserum against the 85-kDa subunit of phosphatidylinositol-3 kinase. Moreover, these growth factors stimulated the association of phosphatidylinositol-3 kinase activity with the Grb2 fusion protein, although this activity was not detected in anti-Shc immunoprecipitates. These results provide further evidence for the divergence of signaling pathways in insulin action, and suggest that Grb2 forms separate complexes with tyrosine-phosphorylated proteins in PC-12 cells.

Cultured rabbit gastric epithelial cells producing prostaglandin I2.

A new method with Dispase, a bacterial neutral protease, was developed for purification of gastric mucosal cells from rabbit fetuses. These cells formed a typical polygonal monolayer after removal of fibroblastlike cells by Dispase treatment, and preserved the features of "normal" cells, exhibiting in vitro aging with a finite life span. The culture consisted mainly of surface mucous cells (50%-60% of the total cell population) and produced prostaglandins (predominantly I2). The addition of acetylsalicylic acid to the culture medium caused marked inhibition of prostaglandin production by the cultured gastric epithelial cells. This epithelial cell strain will be useful in studies on cytoprotection of the stomach.

Actions of exogenous heparan sulfate and hyaluronic acid on growth and thymidine incorporation of normal and transformed human fibroblasts. A comparison with the effects of high cell density and low serum concentration and a warning against thymidine incorporation as a measure of DNA synthesis.

Treatment of normal human (WI-38) cells with exogenous heparan sulfate (HS) reduced cell growth and incorporation of radio-isotope-labeled thymidine (TdR) into DNA. In spite that growth of their transformants (WI-38 CT-1) was enhanced by HS treatment, transformed cells also decreased in TdR incorporation thereby. This peculiar observation was explained by a reduction of TdR uptake, leading to a decrease in specific radioactivity of newly synthesized DNA. The changes in cell growth and TdR incorporation by HS treatment were revealed to be similar to the changes with increasing cell density rather than by serum starvation.

Cytoprotective effect of cyclic AMP on cultured rabbit gastric cells.

A new experimental system with cultured gastric cells was developed to estimate the cytoprotective and damaging effects of drugs. The gastric cells were incubated in vitro in medium of pH 3, and their resistance to acid was determined by measuring percentage of damaged cells as a function of time. Pretreatment with 0.5 mM acetylsalicylic acid (ASA) for 24 h decreased their resistance (26%), whereas pretreatment with 0.3-3 micrograms/ml of 16,16-dimethylprostaglandin E2 (dimethyl PGE2) for 4 h increased their resistance (13-16%). The adenosine 3':5'-cyclic monophosphate (cyclic AMP) level in the cultured gastric cells was decreased by 0.5 mM ASA (30%) and increased by 0.3 microgram/ml of dimethyl PGE2 (38%) on treatment for 24 h. These changes reflect phenomena observed in the gastric mucosa, suggesting that this experimental system is valid as an in vitro model. Concentrations of 0.1 mM cyclic AMP and 0.1 mM N6, O2'-dibutyryladenosine 3':5'-cyclic monophosphate (dibutyryl cyclic AMP) were found to exert cytoprotective effects on the cells (5 and 7% increase, respectively). Furthermore, 0.1 mM cyclic AMP caused partial recovery from 0.5 mM ASA-induced decrease in resistance (ASA: 17%, ASA + cyclic AMP: 9%). These findings suggest that cyclic AMP plays a role in protection of cultured gastric cells and presumably also of gastric mucosal cells in vivo.

Diacylglycerol, but not inositol 1,4,5-trisphosphate, accounts for platelet-derived growth factor-stimulated proliferation of BALB 3T3 cells.

Recently we found that an intracellular event related to phosphatidylinositol 4,5-bisphosphate (PIP2) is crucial for platelet-derived growth factor (PDGF)-induced mitogenesis in fibroblastic cells (Matuoka, K., et al.: Science 239:640-643, 1988). In the present study we examined the mitogenic effects of PIP2 and its hydrolysis products introduced into the cytoplasm of BALB 3T3 cells by micro-injection to confirm the role of PIP2 hydrolysis in PDGF stimulation of cell proliferation. Injection of 1,2-dioleylglycerol (diolein) into serum-deprived quiescent cells induced DNA synthesis with the same time course as that induced by exposure of the cells to PDGF and, in the presence of PDGF, caused no additional increase in the cell population entering S phase. The injection of PIP2, inositol 1,4,5-trisphosphate, or 1,2-dioleylphosphatidic acid into the cells did not induce mitogenesis. Consistent results were obtained in experiments in which the cells were exposed to 1-oleyl-2-acetylglycerol (OAG) and ionomycin; namely, OAG stimulated proliferation of BALB 3T3 cells, but ionomycin did not induce any mitogenesis. Desensitization of the protein kinase C pathway by prolonged exposure of the cells to phorbol ester abolished the induction of cell proliferation by subsequent injection of diolein or exposure to phorbol ester or OAG as well as by PDGF challenge. These findings strongly suggest that activation of the protein kinase C system following formation of diacylglycerol by PIP2 hydrolysis is mainly responsible for the mitogenic action of PDGF on BALB 3T3 cells.