Matrix survival signaling: from fibronectin via focal adhesion kinase to c-Jun NH(2)-terminal kinase.

Most transformed cells have lost anchorage and serum dependence for growth and survival. Previously, we established that when serum is absent, fibronectin survival signals transduced by focal adhesion kinase (FAK), suppress p53-regulated apoptosis in primary fibroblasts and endothelial cells (Ilic et al. 1998. J. Cell Biol. 143:547-560). The present goals are to identify survival sequences in FAK and signaling molecules downstream of FAK required for anchorage-dependent survival of primary fibroblasts. We report that binding of the SH3 domain of p130Cas to proline-rich region 1 of FAK is required to support survival of fibroblasts on fibronectin when serum is withdrawn. The FAK-p130Cas complex activates c-Jun NH2-terminal kinase (JNK) via a Ras/Rac1/Pak1/MAPK kinase 4 (MKK4) pathway. Activated (phospho-) JNK colocalizes with FAK in focal adhesions of fibroblasts cultured on fibronectin, which supports their survival, but not in fibroblasts cultured on collagen, which does not. Cells often survive in the absence of extracellular matrix if serum factors are provided. In that case, we confirm work of others that survival signals are transduced by FAK, phosphatidylinositol 3'-kinase (PI3-kinase), and Akt/protein kinase B (PKB). However, when serum is absent, PI3-kinase and Akt/PKB are not involved in the fibronectin-FAK-JNK survival pathway documented herein. Thus, survival signals from extracellular matrix and serum are transduced by FAK via two distinct pathways.

Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes.

The Ras-dependent activation of mitogen-activated protein (MAP) kinase pathways by many receptors coupled to heterotrimeric guanine nucleotide binding proteins (G proteins) requires the activation of Src family tyrosine kinases. Stimulation of beta2 adrenergic receptors resulted in the assembly of a protein complex containing activated c-Src and the receptor. Src recruitment was mediated by beta-arrestin, which functions as an adapter protein, binding both c-Src and the agonist-occupied receptor. beta-Arrestin 1 mutants, impaired either in c-Src binding or in the ability to target receptors to clathrin-coated pits, acted as dominant negative inhibitors of beta2 adrenergic receptor-mediated activation of the MAP kinases Erk1 and Erk2. These data suggest that beta-arrestin binding, which terminates receptor-G protein coupling, also initiates a second wave of signal transduction in which the "desensitized" receptor functions as a critical structural component of a mitogenic signaling complex.

Cytotrophoblasts infected with a pathogenic human cytomegalovirus strain dysregulate cell-matrix and cell-cell adhesion molecules: a quantitative analysis.

Studies of intrauterine human cytomegalovirus (CMV) infection have shown suppressed replication in the decidua and placenta of strongly seropositive women. Biopsy specimens often contain CMV virion glycoprotein B and DNA in syncytiotrophoblasts and villus core macrophages without productive infection. Focal replication occurs in placentas of women with low to moderate neutralizing antibody titres. Infected cytotrophoblasts downregulate key adhesion and immune molecules required for invasiveness and maternal immune tolerance and reduce matrix metalloproteinase-9 protein and activity, impairing degradation of the extracellular matrix. Here, we used flow cytometry and quantitative RT-PCR analyses to quantify differentiation molecules expressed in freshly isolated cytotrophoblasts purified from placentas at term and differentiating cells infected in vitro with VR1814, a pathogenic clinical strain. Cell surface proteins including E-cadherin, VE-cadherin, HLA-G, and CMV receptors--epidermal growth factor receptor and integrins beta1 and alphavbeta3--were expressed on purified cells, as were integrins alpha9 and beta6, which were not previously studied. Infected cytotrophoblasts dysregulate the levels of particular cell-matrix and cell-cell adhesion proteins and their transcripts. CMV replication in late gestation placentas with considerable reserves could deplete cytotrophoblast progenitors, thereby impairing syncytiotrophoblast development and increasing the risk of virus transmission to fetal blood vessels.

Association of PKC delta and active Src in PMA-treated MCF-7 human breast cancer cells.

Phorbol ester treatment of MCF-7 cells led to the tyrosine phosphorylation and activation of PKC delta. However, through Western blot analysis and in vitro immunecomplex kinase assays, we detected a differential localization of tyrosine-phosphorylated PKC delta and catalytically active PKC delta. Catalytically active PKC delta was concentrated in Triton X-100 solubilized-membrane fractions while tyrosine-phosphorylated PKC delta was localized to the cytosol fraction. Phorbol ester treatment of MCF-7 cells stimulated both the time-dependent in vivo association of Src with PKC delta, evidenced in Src immunoprecipitates by the co-immunoprecipitation of PKC delta, and activation of Src, evidenced in Src immunoprecipitates as an increase in reactivity with a Src antibody (clone 28) reactive only with active Src (dephosphorylated on residue 530) and in Src and PKC delta immunoprecipitates by an increase in Src kinase activity. While our data are consistent with reports in the literature showing the activator/stimulus-dependent tyrosine phosphorylation of PKC delta, our data show that the tyrosine phosphorylation of PKC delta is not essential for kinase activity. These results are the first to demonstrate an in vivo association between PKC delta and active Src in the absence of over-expression of either PKC delta or Src, and support the association of Src and PKC delta towards a physiological function.

Isolation and characterization of human fibroblast tenascin. An extracellular matrix glycoprotein of interest for developmental studies.

We have developed a biochemical method for purifying human tenascin from cultured fibroblasts or the culture medium. The method is a series of biochemical procedures including gel filtration, gelatin gel affinity chromatography and ion-exchange high performance liquid chromatography. The final preparation was identified as tenascin from its immunological cross-reactivity to antibody against chicken tenascin, strong hemagglutination activity which has been reported to be one of the biological functions of chicken tenascin, and from the electron microscopic study demonstrating a six-armed structure. Gel chromatography showed that intact human tenascin has an apparent molecular weight of over one million. Analysis of the purified tenascin with SDS-PAGE under reducing conditions demonstrated that tenascin consists of two kinds of subunits (250K and 190K). We established rat x mouse heterohybridoma cell lines which produce tenascin-specific antibodies. One monoclonal antibody (RCB1) was selected for immunohistochemical study and partially characterized. RCB1 bound native tenascin but not reduced and alkylated tenascin. Immunohistochemistry of normal and neoplastic tissues demonstrated that RCB1 bound the connective tissues surrounding the cancer nests and various normal tissues including interstitium of renal distal tubule, periosteum, endosteum, smooth muscles of digestive tract and media of arteries and arterioles.

A 32-kDa protein associated with phospholipase A2-inhibitory activity from human placenta.

Two monomeric 32-kDa proteins, termed 32K-I (pI 5.8) and 32K-II (pI 5.1), were isolated from human placenta, which was solubilized by a Ca2+-chelator. Only 32K-I was associated with PLA2-inhibitory activity. CNBr peptide mapping indicated that 32K-I was distinct from 32K-II and two 36-kDa proteins, called calpactin I and II or lipocortin II and I, which have been shown to possess PLA2-inhibitory activity. 32K-I bound to PS in a Ca2+-dependent manner. 32K-I was detected in many tissues except brain, cardiac and skeletal muscle.

Spontaneous improvement in a case of C1q nephropathy.

A 17-year-old girl showed mild proteinuria accompanied by hematuria and mild hypocomplementemia. A light microscopic study of the first renal biopsy specimen showed diffuse mild to moderate mesangial proliferation and thickening of the glomerular basement membrane (GBM). An immunofluorescence study showed dominant positive staining (3+) of IgG and C1q in the glomerular mesangium and capillary loop. Staining for C3 and fibrinogen was weak or 1+. Staining for IgA and IgM was negative. Electron-dense deposits were present in the mesangial area and also in the subepithelial, subendothelial, and intramembranous space. Urinary findings improved after dipyridamole treatment. The second renal biopsy, which was performed 5 years later, showed histological improvements, and various pictures of washing-out of deposits were also noted in an electron microscopic study. However, dominant positive staining for IgG and C1q was persistent in an immunofluorescence study. The glomerulopathy of this case belongs in the criteria of neither membranoproliferative glomerulonephritis nor lupus nephritis but could be designated as C1q nephropathy. This is the first report of a histological improvement in C1q nephropathy.

Arginine vasopressin stimulates phospholipid methylation in cultured rat mesangial cells: possible role for PGE2 production.

Incorporation of [3H-methyl] groups into phospholipids and prostaglandin E2 (PGE2) production in cultured rat mesangial cells were examined in the presence and absence of arginine vasopressin (AVP). In cells stimulated with AVP, a rapid increase in the incorporation of [3H-methyl] group into phospholipids was observed within 1 min after stimulation. The [3H-methyl] group present in the phospholipids began to decline 2.5 min after stimulation. The production of PGE2 increased with AVP treatment, and the decline in methylated phospholipids paralleled the release of PGE2 in AVP-stimulated cells. The inhibition of phospholipid methylation by treatment with adenosyl-S-isobutyl mercaptan (SIBA) resulted in a marked decrease in AVP-stimulated PGE2 production. In order to determine the identity of the methylated phospholipids, [3H-methyl] incorporation into phosphatidylethanolamine derivatives was examined. In AVP-stimulated cells, an increase of [3H-methyl] labeled phosphatidylcholine and lysophosphatidylcholine was observed after stimulation with AVP, followed by an apparent increase of [3H-methyl] labeled lysophosphatidylcholine. These findings indicate that AVP stimulates phospholipid methylation in cultured rat mesangial cells and phosphatidylcholine, synthesized by a transmethylation pathway, may be a source for PGE2 production.

Isolated airway exposure to toluene diisocyanate results in skin sensitization.

Toluene diisocyanate (TDI), a highly reactive industrial chemical is a leading cause of occupational asthma in westernized countries. It has also been reported to be a skin sensitizer in mice and guinea pigs although instances of skin sensitivity in humans are rare. It is uncertain if skin-contact is necessary to initiate the dermal sensitization. This study sought to determine if exclusive airway exposure to TDI could result in skin sensitivity. A group of guinea pigs was administered 50 microl of 0.6% TDI intratracheally (it.), another group received intranasal (in.) application of 0.6, 1.2, or 1.8% TDI. Eighty percent (4/5) of the it.-dosed animals, and 92% (11/12) of in.-dosed animals exhibited skin sensitivity. None of 14 control animals gave a positive reaction to patch challenge with TDI. These findings indicate that exclusive exposure of the airways to TDI can result in skin sensitivity and suggest that such events may be possible in TDI workers and should be considered in all workers exposed via the airways to chemical sensitizers.

Experimental study of the maternal effects on tumor immunity of infant mice with C1300 mouse neuroblastoma.

The spontaneous regression of neuroblastoma (NB), one of the most common malignant tumors in childhood, is found to occur in 1% to 2% of patients with NB, especially in young infants. An unexpectedly favorable response to therapy is also noticed in infants suggesting the potential presence of an immune mechanism. Monoclonal C1300-S and C1300A-4 cell lines were established from polyclonal C1300 cells in our laboratory. Adult female A/J mice that had rejected 1 x 10(3) NB cells (C1300S-3) or 1 x 10(5)-10(6) NB cells (C1300A-4) were used as immunized mothers. The immunized mothers with C1300A-4 or C1300S-3 were found to have specific antibodies to C1300S-3 cells by 51Cr release assay of complement dependent cytotoxicity. Newborn mice, 24 hours after birth from immunized or nonimmunized mothers, were inoculated with 1 x 10(3) C1300S-3 NB cells. The same antibody that was assayed in the immunized mothers was detected in this offspring by the antibody-dependent cell cytotoxicity (ADCC). The tumor incidence in the offspring of the immunized mothers was found to be less than that of the offspring of the nonimmunized mothers. This study suggests that the lower tumor incidence in the offspring of immunized mothers compared with offspring of nonimmunized mothers may be attributed to their ADCC activity. Furthermore, the antibody that has the ADCC activity was proven to be immunoglobulin G by a serum absorption test using IgG absorbant. This study offers insight into the relationship between transported mother-infant immunoglobulins and on its potential control of NB.

[An adolescent case of myelodysplastic syndrome following aplastic anemia].

A male with myelodysplastic syndrome (MDS) following aplastic anemia is reported. The patient had been diagnosed as aplastic anemia at 8 years of age, and treated with blood transfusions, anabolic and glucocorticoid steroid hormones. Over a period of subsequent twelve years, he had remission and deterioration. At the age of 21, the patient developed a sudden progression of severe anemia, when his bone marrow showed hyperplasia with prominent dyshematopoiesis and excess of blasts, compatible with MDS by the definition of FAB classification. He received low dose Ara-C therapy, which was ineffective. Nine months later he developed acute monocytic leukemia (M5b) and died. Chromosomal analysis revealed 46, XY at the onset of aplastic anemia, 46, XY, del (6) (q21 q27) at the MDS and 46, XY, -7, +21, 6q-/47, XY, +Y, -7, +21, 6q- at the acute leukemic stage.

Platelet-activating factor induces cell growth through tyrosine phosphorylation pathway in cultured rat mesangial cells.

Accumulating evidence suggests that platelet-activating factor (PAF) may play a role in renal pathophysiology. Therefore, in order to investigate this notion further, the effects of PAF on cell growth and tyrosine phosphorylation were analyzed in cultured rat mesangial cells. PAF was found to enhance a time and concentration-dependent increase in phosphotyrosine in several proteins and stimulate 3H-thymidine incorporation. Tyrosine phosphorylation was also enhanced by PAF in protein kinase C (PKC) depleted cells, whereas a tyrosine kinase inhibitor, genistein, inhibited tyrosine phosphorylation of these proteins at the concentration of 1 microgram/ml. PAF stimulated 3H-thymidine incorporation at concentrations below 10(-6) M, but exerted progressive inhibition at concentrations above 10(-6) M. Pre-treatment with phorbol 12-myristate 13-acetate (PMA) did not affect PAF-enhanced incorporation at lower concentrations of PAF, and reversed the inhibitory effects of PAF at higher concentrations. Finally, genistein pre-treatment completely inhibited PAF-induced cell growth at the concentration of 1 microgram/ml. Both tyrosine phosphorylation and 3H-thymidine incorporation induced by PAF were completely inhibited by pre-treatment with the PAF-receptor antagonist, CV-6209, at the concentration of 10(-5)M. These results suggest that PAF enhancement of tyrosine phosphorylation occurred in a PKC-independent manner and that a tyrosine kinase was associated with PAF-induced tyrosine phosphorylation. Moreover, they indicate that the phosphoinositide hydrolysis-PKC pathway is not essential for PAF-induced cell proliferation, and that PKC activation may play an inhibitory rather than a stimulatory role in mitogenesis in response to PAF. Our results indicate that the tyrosine phosphorylation pathway induced by PAF may participate critically in downstream mitogenic signaling through the PAF receptor.

[A case of rapidly progressive IgA nephropathy with transient hypocomplementemia at onset].

We present a case of IgA nephropathy (IgAGN) which developed rapidly progressive glomerulonephritis and showed marked clinical improvement with treatment. The patient was a 7-year-old boy who initially presented with acute nephritic syndrome with hypocomplementemia. Although the renal function improved with normalization of the serum complement level, it deteriorated again progressively. The first renal biopsy revealed cellular crescents in about 70 percent of 43 glomeruli. Immunofluorescent microscopy demonstrated deposits of IgA, C3 and IgG in the mesangium; they were also deposited along the glomerular capillary walls. He was treated with plasma exchange associated with hemodialysis and methylprednisolone pulse therapy, followed by oral administration of prednisolone, cyclophosphamide and warfarin. Renal function recovered to the normal range about two months after the initiation of treatment. The second biopsy demonstrated a marked decrease in histological activity. In this case, transient hypocomplementemia at onset may indicate that acute glomerulonephritis caused exacerbation of clinically silent IgAGN. Aggressive therapy may be effective in patients with rapidly progressive IgAGN if treated at an early stage.

Real-time observation of FIB-created dots and ripples on GaAs.

We report a phenomenological study of Ga dots and ripples created by a focused ion beam (FIB) on the GaAs(001) surface. Real-time observation of dot diffusion and ripple formation was made possible by recording FIB movies. In the case of FIB irradiation with a 40 nA current of Ga(+) ions accelerated under 40 kV with an incidence angle of θ = 30°, increasing ion dose gives rise to three different regimes. In Regime 1, dots with lateral sizes in the range 50-460 nm are formed. Dots diffuse under continuous sputtering. In Regime 2, dots self-assemble into Bradley and Harper (BH) type ripples with a pseudo-period of λ = 1150 ± 25 nm. In Regime 3, ripples are eroded and the surface topology evolves into microplanes. In the case of normal incidence, FIB sputtering leads only to the formation of dots, without surface rippling.

Activated c-SRC in ductal carcinoma in situ correlates with high tumour grade, high proliferation and HER2 positivity.

Overexpression and/or activity of c-Src non-receptor tyrosine kinase is associated with progression of several human epithelial cancers including breast cancer. c-Src activity in 'pure' ductal carcinoma in situ (DCIS) was measured to assess whether this predicts recurrence and/or correlates with HER2 expression and other clinical parameters. Activated c-Src levels were evaluated in DCIS biopsies from 129 women, with median follow-up at 60 months. High levels of activated c-Src correlated with HER2 positivity, high tumour grade, comedo necrosis and elevated epithelial proliferation. In univariate analysis, high activated c-Src level associated with lower recurrence-free survival at 5 years (P=0.011). Thus, high c-Src activity may identify a subset of DCIS with high risk of recurrence or progression to invasive cancer where therapeutics targeting c-Src may benefit this patient subset.

Hydroxyapatite-pulp composite fiber sheet bed: A new material for the immobilization of CHO-K1 cells.

A new immobilization material for cell culture, ahydroxyapatite-pulp composite fiber (HAPC) sheet bed, was usedto grow CHO-K1 cells. The sheet bed for cell culture wasprepared from HAPC fiber by paper-making techniques. Scanning electron microscopic analysis revealed that the HAPCsheet bed had a structure consisting of piled fibers with spaces 10-200 mum in diameter and a pore surface area of 0.32 m(2) g(-1). Using a 25 x 25 mm(2) squareHAPC sheet bed 0.41 mm in thickness (85 g m(-2) basis weight) for cell culture, CHO-K1 cells grew to a cell densityof 3.7 x 10(7) cells cm(-3) in a 60 mm plastic dish over a 6-day culture period. High-density culture of CHO-K1 cells was successfully performed using the HAPC sheet bed in a 500 ml spinner flask over a 21-day culture period. The HAPC sheet bed, wound around the stirrer paddle, was rotated in the spinner flask in order to supply nutrientsand remove waste products efficiently. The HAPC sheet bedhas a large surface area to support cell growth and there islarge diffusion space inside of the bed. This newautoclavable substrate for anchorage-dependent cells can be easily scaled-up.