FAK-Src-paxillin system expression and disease outcome in human neuroblastoma.

BACKGROUND: Neuroblastoma (NB) often presents with metastatic disease and poor survival. The need for new prognostic markers remains invaluable. The FAK-Src-Paxillin protein system is associated with aggressive phenotype in adult malignancies but is largely unexplored in pediatric NB. OBJECTIVE: To assess FAK-Src-Paxillin protein expression in human NB cell lines and clinical cytology material and to delineate its association with survival. DESIGN/METHODS: Western blot and immunohistochemistry were applied for FAK-Src-Paxillin expression in NB cell lines and 23 human cytology specimens, respectively. Protein expression in human clinical samples was correlated with clinicopathological parameters, MYCN amplification and survival. RESULTS: FAK, Src and Paxillin proteins are expressed in human NB cells lines, and can be detected in clinical cytology specimens from NB patients, (59%, 32% and 33% respectively). Simultaneous FAK-Src-Paxillin expression was noted in 30% of NB patients. Children with concomitant positivity FAK, Src, and Paxillin tumors, as well as MYCN amplification, had increased mortality compared to those without. CONCLUSIONS: FAK-Src-Paxillin system is a marker of unfavorable prognosis for human NB patients but also a promising therapeutic target.

Problems encountered in bicistronic IRES-GFP expression vectors employed in functional analyses of GC-induced genes.

Our laboratory has developed a series of Gateway(®) compatible lentiviral expression systems for constitutive and conditional gene knock-down and over-expression. For tetracycline-regulated transgenic expression, we constructed a lentiviral "DEST" plasmid (pHR-TetCMV-Dest-IRES-GFP5) containing a tetracycline-responsive minimal CMV promoter, followed by an attP site-flanked DEST cassette (for efficient cloning of cDNAs by "Gateway(®)" recombination cloning) and green fluorescent protein (GFP) driven by an internal ribosomal entry site (IRES).This lentiviral bicistronic plasmid allows immediate FACS identification and characterization of successfully transfected cell lines. Although this system worked well with several cDNAs, we experienced serious problems with SLA, Bam and BMF. Particularly, we cloned the cDNA for human SLA (Src-like adapter), a candidate gene in GC-induced apoptosis, into this plasmid. The resulting construct (pHR-TetCMV-SLA-IRES-GFP5) was transfected into HEK 293-T packaging cells to produce viral particles for transduction of CEM-C7H2-2C8 cells. Although the construct produced many green fluorescent colonies at the HEK 293-T and the CEM-C7H2-2C8 level, we could not detect any SLA protein with α-SLA antibody from corresponding cell lysates. In contrast, the antibody readily detected SLA in whole cell lysate of HEK 293-T cells transfected with a GST-flagged SLA construct lacking IRES-GFP. To directly address the potential role of the IRES-GFP sequence, we cloned the SLA coding region into pHR-TetCMV-Dest, a vector that differs from pHR-TetCMV-Dest-IRES-GFP5 just by the absence of the IRES-GFP cassette. The resulting pHR-TetCMV-SLA construct was used for transfection of HEK 293-T cells. Corresponding lysates were assayed with α-SLA antibody and found positive. These data, in concert with previous findings, suggest that the IRES-GFP cassette may interfere with translation of certain smaller size cDNAs (like SLA) or generate fusion proteins and entail defective virus production in an unpredictable manner.

Dasatinib (BMS-35482) has synergistic activity with paclitaxel and carboplatin in ovarian cancer cells.

PURPOSE: To explore the activity of dasatinib alone and in combination with paclitaxel and carboplatin in ovarian cancer cells and to determine if dasatinib activity can be predicted based on evaluation of the SRC pathway. EXPERIMENTAL DESIGN: Microarray analysis was performed for IGROV1, OVCAR3, A2780 and SKOV3 ovarian cancer cells and the status of the genomic SRC signature pathway was determined. Cells were treated with carboplatin, paclitaxel and dasatinib individually and in combination. Pre- and post-treatment phospho-SRC (pSRC) and SRC protein expression was determined. Dose-response curves were constructed, and drug interaction was assessed by the Combination Index (CI) method. RESULTS: SRC protein expression levels reflected the SRC pathway genomic signature in the cell lines with the lowest (SKOV3) and highest (IGROV1) pathway expression, but not in those with intermediate expression (OVCAR3, A2780). Dasatinib treatment caused loss of pSRC in all cell lines, with 50% growth inhibition for IGROV1 at 70 nM, OVCAR3 at 34 nM, A2780 at 4.1 µM and SKOV3 at 530 nM. Dasatinib combined with cytotoxics yielded a synergistic effect (CI=0.46 to 0.79) in all cell lines except SKOV3. CONCLUSION: Dasatinib in combination with standard chemotherapeutic agents appears to interact in a synergistic manner in some ovarian cancer cell lines. Further research is needed to evaluate tumor cell characteristics which predict response to dasatinib.

Osteoclasts express high levels of pp60c-src in association with intracellular membranes.

Deletion of the c-src gene in transgenic mice by homologous recombination leads to osteopetrosis, a skeletal defect characterized by markedly deficient bone resorption (Soriano, P., C. Montgomery, R. Geske, and A. Bradley. 1991. Cell. 64:693-702), demonstrating a critical functional role of pp60c-src in osteoclast activity. Since decreased bone resorption could result from a defect either within the osteoclast or within other cells present in its environment, indirectly affecting osteoclast functions, we determined which cell(s) in bone expressed high levels of pp60c-src Measuring pp60c-src protein and kinase activities in osteoclasts and immunolocalizing pp60c-src in bone, we find that expression of pp60c-src is nearly as high in osteoclasts as in brain and platelets. In contrast, other bone cells contain only very low levels of the protein. In addition, expression of the c-src gene product increases when bone marrow cells are induced to express an osteoclast-like phenotype by 1,25-dihydroxy-vitamin D3, further suggesting that high expression of pp60c-src is part of the osteoclast phenotype. Three other src-like kinases, c-fyn, c-yes, and c-lyn, are also expressed in osteoclasts at ratios to pp60c-src similar to what is found in platelets. These src-related proteins do not, however, compensate for the absence of pp60c-src in the src- mice, thereby suggesting that pp60c-src may have a specific function in osteoclasts. Although further work is necessary to elucidate what the critical role of pp60c-src in osteoclasts is, our observation that the protein is associated mostly with the membranes of intracellular organelles suggests the possibility that this role might be at least in part related to the targeting or fusion of membrane vesicles.

PP2 regulates human trophoblast cells differentiation by activating p38 and ERK1/2 and inhibiting FAK activation.

Throughout gestation, fetal growth and development depend, in part, on placental transfer of nutrients from the maternal circulation. This latter function depends on multinucleated, terminally differentiated syncytiotrophoblasts. In vitro, freshly isolated cytotrophoblast cells differentiate spontaneously into syncytiotrophoblast in the presence of fetal bovine serum (FBS). We have previously showed that trophoblast differentiation is regulated by ERK1/2 and p38. Moreover, we showed that PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3, 4-d]pyrimidine], a Src family kinase (SFK) specific inhibitor, stimulates biochemical trophoblast cells differentiation while it inhibits cell adhesion and spreading without affecting cell fusion. Therefore, we examined the mechanisms by which PP2 modulates trophoblast cells differentiation. This study shows that PP2 stimulates ERK1/2 and p38 activation after 24h of treatments and up to 3 days while it inhibits focal adhesion kinase (FAK) phosphorylation at many sites including Tyr-397, 407, 576 and 577. Furthermore, we showed that transient activation of ERK1/2 by FBS is independent of SFK and that PP2 induces rapid activation of p38. Moreover, the kinase activity of SFK is negatively regulated by the phosphorylation of their carboxy (C)-terminal regulatory tyrosines by specific proteins called carboxyl-terminal Src kinase (Csk) and Csk homologous kinase (CHK). We showed the expression of Csk and CHK in human trophoblast cells. In summary, this study showed that PP2 stimulates the biochemical differentiation of trophoblast cells by stimulating p38 and ERK1/2 while it inhibits the morphological differentiation by inhibiting FAK activation.

The cell cycle and c-Src.

The activity of the proto-oncogene encoded c-Src product is tightly regulated in vivo. In recent years, a model has emerged of how this regulation is achieved. In particular, protein kinases and phosphatases that are potential regulators of c-Src activity in the cell cycle have been identified and characterized.

The nonreceptor protein-tyrosine kinase CSK complexes directly with the GTPase-activating protein-associated p62 protein in cells expressing v-Src or activated c-Src.

CSK is a predominantly cytosolic protein-tyrosine kinase (PTK) that negatively regulates Src family PTKs by phosphorylation of a conserved tyrosine near their C termini. Little is known about how CSK itself is regulated. On the basis of immunofluorescence studies, a model has been proposed that when c-Src is activated, it is redistributed to podosomes, in which substrates become phosphorylated, creating binding sites for CSK. CSK is recruited to these sites of c-Src activation via its SH2 and SH3 domains and is then in a position to downregulate c-Src activity (B. W. Howell and J. A. Cooper, Mol. Cell. Biol. 14:5402-5411, 1994). To identify phosphotyrosine (P.Tyr)-containing proteins that may mediate translocation of CSK due to c-Src activation, we have examined the whole spectrum of P.Tyr-containing proteins that associate with CSK in v-Src NIH 3T3 cells by anti-P.Tyr immunoblotting. Nine P.Tyr-containing proteins coimmunoprecipitated with CSK from v-Src NIH 3T3 cells. One of these, an approximately 62-kDa protein, also associated with CSK in NIH 3T3 cells treated with vanadate prior to lysis and in NIH 3T3 cells expressing an activated c-Src mutant. This 62-kDa protein was shown to be identical to the GTPase-activating protein (GAP)-associated p62 (GAP-A.p62) protein. The interaction between CSK and GAP-A.p62 could be reconstituted in vitro with glutathione S-transferase fusion proteins containing full-length CSK or the CSK SH2 domain. Furthermore, our data show that CSK interacts directly with GAP.A-p62 and that the complex between the two proteins is localized in subcellular membrane or cytoskeletal fractions. Our results suggest that GAP-A.p62 may function as a docking protein and may mediate translocation of proteins, including GAP and CSK, to membrane or cytoskeletal regions upon c-Src activation.

Carboxyl-terminal Src kinase homologous kinase negatively regulates the chemokine receptor CXCR4 through YY1 and impairs CXCR4/CXCL12 (SDF-1alpha)-mediated breast cancer cell migration.

Using microarray gene analysis, we found that carboxyl-terminal Src kinase homologous kinase (CHK) regulated the expression of the chemokine receptor, CXCR4. Northern blot and fluorescence-activated cell-sorting analyses showed that CHK down-regulated CXCR4 mRNA and protein levels, respectively. Mutated CHK, which contains a mutation within the ATP binding site of CHK, failed to inhibit CXCR4 expression, thus suggesting that CHK kinase activity is involved in the regulation of CXCR4. Results from gel shift analysis indicated that CHK regulates CXCR4 transcriptional activity by altering YY1 binding to the CXCR4 promoter. Whereas CHK had no significant effects on the expression of YY1, c-Myc, Max, and other YY1-binding proteins, CHK was found to modulate the YY1/c-Myc association. Furthermore, CHK inhibited CXCR4-positive breast cancer cell migration. Taken together, these studies show a novel mechanism by which CHK down-regulates CXCR4 through the YY1 transcription factor, leading to decreased CXCR4-mediated breast cancer cell motility and migration.

CHK negatively regulates Lyn kinase and suppresses pancreatic cancer cell invasion.

Among the most important signaling pathways operating in pancreatic cancer cells are those resulting from mutations in the Ras oncogene or from overexpression of ErbB-2 and associated Src-family kinases. In this study, we aimed to characterize CHK expression and function in pancreatic cancer. Our data demonstrated CHK expression in human pancreatic cancer tissues, and also showed that CHK associated with ErbB-2 via its SH2 domain in human PANC-1 pancreatic cancer cells. PANC-1 cells were found to express both Src kinase and Lyn kinase, although the expression of Lyn kinase was more abundant. Furthermore, CHK downregulated Lyn kinase activity and significantly inhibited the in vitro growth and invasion of PANC-1 cells upon EGF stimulation. These results indicate that CHK is a negative regulator of ErbB-2 and Lyn kinase signaling in pancreatic cancer cells.

Mutational activation of pp60(c-src) leads to a tumorigenic phenotype in a preneoplastic Syrian hamster embryo cell line.

Previous studies indicated that overexpression of wild-type avian c-src cannot induce neoplastic transformation of NIH 3T3 cells. In this study, we isolated and characterized novel spontaneously derived transforming mutants of avian pp60(c-src) from a Syrian hamster embryo-derived cell line, 10W, transfected with the avian c-src gene. Seventeen independently derived transfected 10W cell clones were injected into athymic nude mice. After a latency period, tumors eventually arose and were established in culture. The tumorigenic phenotype was always accompanied by the presence of the avian c-src DNA and functional expression of pp60(c-src). However, most of the tumor-derived cell lines expressed an electrophoretically altered form of pp60(c-src), suggesting mutations in src. Consistent with this hypothesis, DNAs isolated from the tumor-derived lines, but not the parental 10W cell lines, morphologically transformed NIH 3T3 cells in a focus-forming assay. We characterized pp60(c-src) in detail from three of the tumor-derived lines: 4AT, 4BT, and E2T. Two of these lines contained mutations within the exogenous c-src coding region. Line 4AT has an internal repeat of 29 amino acids immediately following Gln-513, which disrupts the spacing between the end of the kinase domain and Tyr-527, the negative regulatory site in pp60(c-src). Line 4BT has a 5-bp deletion following Phe-520, which results in loss of Tyr-527. However, the DNA sequence of the coding region of pp60(c-src) from a third line, E2T, was completely wild type. Cyanogen bromide cleavage analyses of the altered pp60(c-src) from lines 4AT and 4BT showed that Tyr-527, the site of negative regulation of c-src, is not phosphorylated, but Tyr-416, the site of in vitro autophosphorylation, is phosphorylated. However, in line E2T, Tyr-527 was phosphorylated, and Tyr-416 was phosphorylated to a lesser extent. Additionally, two proteins that indicate activation of src, p85 cortactin and p120(cas), are phosphorylated in at least six of the tumor-derived cell lines, although to a lesser extent in line E2T. These results suggest that dephosphorylation of Tyr-527 and phosphorylation of Tyr-416 correlate with activation of pp60(c-src) in the tumor-derived lines 4AT and 4BT, respectively. However, in line E2T, the high levels of pp60(c-src), in combination with a partial activation of the pp60(c-src) protein as indicated by phosphorylation of Tyr-416, appear to be involved in the neoplastic process, rather than mutation.

V-SRC induces expression of hypoxia-inducible factor 1 (HIF-1) and transcription of genes encoding vascular endothelial growth factor and enolase 1: involvement of HIF-1 in tumor progression.

Adaptation to hypoxia represents an important aspect of tumor progression. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that mediates essential homeostatic responses to cellular and systemic hypoxia by activating transcription of multiple genes including those encoding glycolytic enzymes and vascular endothelial growth factor (VEGF). In this report, we demonstrate that whereas C-SRC expression is not required for expression of HIF-1 or transcriptional activation of genes encoding VEGF and enolase 1 (ENO1), cells expressing the v-Src oncogene have increased expression of HIF-1, VEGF, and ENO1 under both hypoxic and nonhypoxic conditions. Expression of V-SRC was associated with increased transcription of reporter genes containing cis-acting hypoxia-response elements from the VEGF and ENO1 genes, and this transcriptional activation required an intact HIF-1 binding site. When three rat hepatoma subclones that differed with respect to the level of HIF-1 expression were injected into nude mice, tumor growth correlated with HIF-1 expression, suggesting that HIF-1 may be generally involved in tumor progression. These studies link an oncogene to the induction of HIF-1 expression, thus providing a mechanism for hypoxic adaptation by tumor cells.

Effect of herbimycin A on growth and pp60c-src activity in human colon tumor cell lines.

The effect of herbimycin A, an ansamycin antibiotic which inhibits cellular transformation by retroviral tyrosine kinases, on the monolayer growth of seven colon tumor cell lines and one cell line established from normal colonic mucosa, CCL239, was examined. Each colon tumor cell line tested showed dose-dependent growth inhibition in response to herbimycin A. A 125ng ml-1 dose of the antibiotic caused greater than 40% growth inhibition in all colon tumor cell lines after two cell doublings. In contrast, at similar herbimycin A concentrations only 12% inhibition was observed in 'normal' CCL239 cells. No major morphologic changes were observed at the light microscopic level in any of the tumor cell lines or CCL239 cells in response to treatment with herbimycin A. Studies using the HT29 colon adenocarcinoma cell line showed dose-dependent inactivation of pp60c-src by herbimycin A, resulting in decreased autophosphorylation, enolase phosphorylation and steady-state levels, which correlated with cellular growth inhibition. Herbimycin A-induced reductions in pp60c-src kinase activity preceded changes in pp60c-src steady-state levels. Growth and pp60c-src inhibition were reversible following removal of herbimycin A from cell culture media. Our results suggest that regulation of pp60c-src tyrosine kinase activity may be important in growth control of colon tumor cells.

Inhibition of pp60c-Src reduces Bcl-XL expression and reverses the transformed phenotype of cells overexpressing EGF and HER-2 receptors.

Tumors that overexpress HER-2/neu receptor or exhibit enhanced EGFR signaling have been reported to possess constitutively activated Src family kinases, especially pp60c-Src. High levels of pp60c-Src activity have also been reported for cell lines that overexpress the EGFR or the chimeric EGFR-HER-2 receptor. It has therefore been suggested that Src kinases may contribute significantly to the oncogenic phenotype of these cells and to the degree of malignancy of tumors that overexpress EGFR family receptors. In this study we show that the induced expression of c-SRC antisense RNA or the application of a selective Src kinase inhibitor induces growth arrest, programmed cell death and reverses the transformed properties of cells that overexpress EGFR or HER-2 receptors. We show that inhibition of Src kinase expression or activity results in the reduction of Stat3 tyrosine phosphorylation, decline of Bcl-XL expression, and induction of cell death. Using a construct in which the promoter of Bcl-X, which possesses putative Stat3 sites, is tethered to the luciferase reporter gene, we show that inhibition of Src activity or expression induces a decline in Bcl-X expression. We also show that the expression of activated Src induces activation of the Bcl-X promoter. This activation is inhibited by the expression of kinase dead Src or of Stat3beta, the dominant-negative form of Stat3. Taken together, these results support the hypothesis that Src positively regulates the transformed phenotype of cells overexpressing EGFR family kinases. Furthermore, these results also suggest that Src positively regulates Bcl-XL expression via Stat3 activation and thus acts not only as a potent mitogenic signaling element, but also as an anti-apoptotic signaling protein. The combination of both activities probably confers upon activated Src its oncogenic activity. Since Src kinase is activated in many tumors, pp60c-Src kinase inhibitors may prove useful as anti-cancer agents for many types of cancer.

Oncogene-mediated transformation of fetal rat colon in vitro.

Short-term maintenance of fetal rat colonic tissue in vitro has been demonstrated using a collagen matrix organ culture system. The introduction of single (v-myc, v-rasH, v-src) oncogenes or combinations of oncogenes (v-myc/rasH, v-myc/src) into normal colon mucosal elements was established using retroviral vectors, resulting in enhanced proliferation and migration of epithelial cells from the lumen of tissue implants. Expression of a single oncogene in normal colon epithelium did not result in the establishment of cell lines. In contrast, expression of cooperating oncogenic elements resulted in cell lines in greater than 80% of experiments, revealing different morphological characteristics dependent upon the oncogene combination used. Confirmation of the expression of viral transcripts was determined using Northern blot analysis and viral oncoprotein expression using Western blot analysis (p21) and an immunoprecipitation kinase assay (src). Expression of keratin filaments was lost following passaging of cell lines but could be induced by the myc/ras transformants by growth on Rat-1 feeder layers. This induction phenomenon was not observed with myc/src lines, and although these expressed high levels of sucrase isomaltase the epithelial origin of these cells is unclear. Karyotypic analysis performed on three myc/ras-transformed cell lines revealed a normal chromosome complement associated with transformation. In this report we describe a novel in vitro transformation system for normal rat colonic epithelium mediated by the introduction of oncogene elements using different retroviral vector constructs. The potential to generate cell lines representing different stages of neoplastic progression using relevant genetic components presents significant advantages for the study of cellular and molecular interactions underlying colon neoplastic progression.

Novel insights into c-Src.

Since identifying a transmissible agent responsible for tumorigenesis in chickens, the v-Src oncogene, significant progress has been made in determining the functions of its cellular homologue. c-Src is the product of the SRC gene and has been found both over-expressed and highly activated in a number of human cancers. In fact the relationship between c-Src activation and cancer progression is significant. Furthermore c-Src may play a role in the acquisition of the invasive and metastatic phenotype. In this review we will summarize some of the latest evidence for the role of c-Src in tumorigenesis and particularly in human tumor progression. In this review, specifically, we will address growth signals, adhesion, migration, invasion, angiogenesis and functional genomics.

Enhanced expression of N-myc messenger RNA in neuroblastomas found by mass screening.

A substantial fraction of neuroblastomas found by mass screening have been suggested to regress spontaneously because of the high incidence of infantile neuroblastomas in the screening population. In this study, 70 neuroblastomas were analyzed for expression of proto-oncogenes related to neuronal differentiation to clarify the biological significance of proto-oncogene expression in the screening-positive and -negative tumors. The tumors consisted of 39 neuroblastomas found by screening (group 1), 16 non-N-myc-amplified neuroblastomas found by clinical symptom(s) (group 2), and 15 N-myc-amplified neuroblastomas found by clinical symptom(s) (group 3). The expression of c-src, trk A, and N-myc in tumor tissues was analyzed by quantitative RNA PCR. Neuronal c-srcN2 expression varied significantly in the following order: group 1 > group 2 > group 3. The level of expression of trk A was markedly reduced in group 3 but did not differ in groups 1 and 2. Most tumors in group 3 overexpressed N-myc. However, N-myc expression in group 1 was significantly higher than that in group 2. Thus, the characteristics of proto-oncogene expression in screening-positive tumors included enhanced expression of c-srcN2 and N-myc mRNA, regardless of nonamplification of N-myc. Our results suggest that the role of N-myc differs in neuroblastomas detected by screening and in N-myc-amplified tumors.

Berberine reduces Toll-like receptor-mediated macrophage migration by suppression of Src enhancement.

Berberine is an isoquinoline with anti-inflammatory activity. We previously demonstrated that there was a loop of signal amplification between nuclear factor kappa B and Src for macrophage mobility triggered by the engagement of Toll-like receptors (TLRs). The simultaneous suppression of lipopolysaccharide (LPS)-mediated upregulation of inducible nitric oxide synthase, cyclooxygenase 2, and cell mobility in berberine-treated macrophages suggested Src might be a target of berberine. Indeed, th reduced migration, greatly suppressed Src induction in both protein and RNA transcript by berberine were observed in macrophages exposed to LPS, peptidoglycan, polyinosinic-polycytidylic acid, and CpG-oligodeoxynucleotides. In addition to Src induction, berberine also inhibited LPS-mediated Src activation in Src overexpressing macrophages and S-nitroso-N-acetylpenicillamine (a nitric oxide donor) could partly restore it. Moreover, berberine suppressed Src activity in fibronectin-stimulated macrophages and in v-Src transformed cells. These results implied that by effectively reducing Src expression and activity, berberine inhibited TLR-mediated cell motility in macrophages.

Osteoclasts express high levels of p60c-src, preferentially on ruffled border membranes.

Expression of p60c-src, the normal cellular counterpart of the transforming protein of Rous sarcoma virus (RSV), p60v-src, was examined in mouse and rat authentic osteoclasts and mouse osteoclast-like multinucleated cells (MNCs) formed in vitro. In co-cultures of mouse osteoblastic cells and spleen cells, the expression of p60c-src strikingly increased on day 5 in parallel with the appearance of MNCs in the presence of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3). Immunohistochemical examination confirmed the high level expression of p60c-src in both mouse authentic osteoclasts and MNCs. Electron microscopic examination revealed that p60c-src was primarily localized on ruffled border membranes and vacuoles, but not on the clear zone in rat authentic osteoclasts. These results suggest that p60c-src is important in osteoclastic bone resorption.

Interferon-gamma and tumor necrosis factor-alpha enhance p60src expression in human macrophages and myelomonocytic cell lines.

We investigated modulation of p60src expression in human mononuclear phagocytes. By analysis of [35S]methionine-labelled cells we found that synthesis of p60src is higher in human monocytes compared to macrophages derived from in vitro cultivation of monocytes. Western blot analysis showed that expression of p60src in monocyte-derived macrophages can be enhanced if monocytes are differentiated into macrophages in the presence of interferon-gamma (IFN-gamma), or tumor necrosis factor-alpha (TNF-alpha). Enhanced p60src expression caused by IFN-gamma or TNF-alpha correlated with an enhanced autophosphorylating kinase activity assayed in anti-p60src immune precipitates. In vivo phosphorylation of p60src and analysis of phosphopeptides by tryptic digestion showed that treatment with cytokines did not affect the pattern of phosphorylation of distinct phosphopeptides. The human monocytic cell lines, U937 and HL-60, induced to differentiate along the monocytic pathway by IFN-gamma, or a combination of IFN-gamma and TNF-alpha, expressed higher amounts of the p60src, but not of the p59fyn or p62yes, kinase activity. These findings show that p60src is modulated in the course of differentiation of human monocytes to macrophages, and that macrophage-activating cytokines increase p60src expression in human monocyte-derived macrophages.

Uni-axial cyclic stretch induces c-src activation and translocation in human endothelial cells via SA channel activation.

The kinase activity of c-src increased and peaked at 15 min after an application of uni-axial cyclic stretch in HUVECs followed by a translocation of c-src to Triton-insoluble fraction. Suppression of c-src by an antisense S-oligodeoxynucleotide inhibited the stretch-induced tyrosine phosphorylation and morphological changes. The stretch-induced increase in c-src activity was inhibited by FK506, a specific inhibitor for calcineurin, by Gd3+, a blocker for stretch activated channels, and by the extracellular Ca2+ depletion suggesting the involvement of SA channels. These results strongly suggest c-src plays an important role in the downstream of SA channel activation followed by the morphological changes.