PubMed Health⌕ Search

Biomedical subjects

A Gazit

Publications and source records attributed to A Gazit.

At least 19 recordsLinked to original sources
PubMed2001

Constitutive activation of Stat3 by the Src and JAK tyrosine kinases participates in growth regulation of human breast carcinoma cells.

Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological effects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our findings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.

Animals↗
PubMed2001

The cytoskeleton-associated TCR zeta chain is constitutively phosphorylated in the absence of an active p56(lck) form.

The TCR recognizes peptide-MHC complexes and transmits activation signals leading to cellular responses. We have previously characterized two TCR populations expressed on the T cell surface; one is linked to the cytoskeleton via a detergent-insoluble cytoskeleton-associated zeta (cska-zeta) chain, while the other is detergent soluble and not linked to the cytoskeleton. The cska-zeta form displays unique properties: it is constitutively phosphorylated, does not undergo hyperphosphorylation upon TCR stimulation as opposed to its non-cytoskeleton-associated counterpart (non-cska-zeta) and it maintains a molecular mass of 16 kDa. It is well established that p56(lck) and possibly p59(fyn) are responsible for the generation of the 21/23-kDa phosphorylated detergent-soluble zeta form. We now demonstrate that the phosphorylation of cska-zeta does not require the activity of p56(lck). We also show that although Lck does not phosphorylate cska-zeta in vivo, it retains the capacity to phosphorylate cska-zeta in vitro. Moreover, differences in zeta-associated kinase activity were detected for non-cska-zeta and cska-zeta. Our results indicating that different kinases phosphorylate the two zeta forms are consistent with a growing consensus that each TCR form may regulate distinct cellular functions.

Animals↗
PubMed2001

Potential (18)F-labeled biomarkers for epidermal growth factor receptor tyrosine kinase.

As PET candidate tracers for EGFr-TK, five 4-(anilino)quinazoline derivatives, each fluorinated in the aniline moiety, were prepared. Each was tested in vitro for inhibition of EGFr autophosphorylation in A431 cell line. The leading compounds were then radiolabeled with (18)F and cell binding experiments, biodistribution and PET studies in A431 tumor-bearing mice were performed. Metabolic studies were carried out in a mice control group. From our results, we concluded that while in vitro experiments indicates efficacy of 4-(anilino)quinazoline compounds, kinetic factors and rapid blood clearance make them unsuitable as tracers for nuclear medicine imaging of EGFr-TK.

Animals↗
PubMed2001

Novel mechanism of Wnt signalling inhibition mediated by Dickkopf-1 interaction with LRP6/Arrow.

Wnt signalling has an important role in cell fate determination, tissue patterning and tumorigenesis. Secreted antagonists of Wnt include Frizzled (Fz)-related proteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (Dkk). FRPs, Cerberus and WIF have all been shown to act by binding and sequestering Wnt. We report a novel mechanism of Wnt-signalling inhibition by human Dkk-1. Dkk-1 demonstrated no interaction with Wnt but bound a single cell surface site with high affinity (K(D) = 0.39 nM). Its receptor was detectable in a complex with a relative molecular mass of 240,000 (M(r) 240K) with [(125)I] Dkk-1 by covalent affinity cross-linking. Wnt signalling through beta-catenin is mediated by the Fz receptor and a recently identified low-density-lipoprotein-receptor-related co-receptor, LRP6/Arrow. Overproduction of the 200K LRP6 protein, but not of Fz, strikingly increased Dkk-1 binding as well as the amount of the 240K cross-linked complex, which was shown to be composed of Dkk-1 and LRP6. Moreover, Dkk-1 function was completely independent of Fz but LRP6 dramatically interfered with the Dkk-1 inhibition of Wnt signalling. Thus, unlike Wnt antagonists, which exert their effects by molecular mimicry of Fz or Wnt sequestration through other mechanisms, Dkk-1 specifically inhibits canonical Wnt signalling by binding to the LRP6 component of the receptor complex.

Cell Line↗
PubMed2000

Substrate competitive inhibitors of IGF-1 receptor kinase.

IGF-1 and its receptor play a pivotal role in many cancers, and therefore, IGF-1R is an attractive target for the design of inhibitors. In this communication, we report on a number of lead compounds for inhibitors of the isolated IGF-1R kinase. The search for these compounds utilized two novel in vitro assays and was aided by the knowledge of the three-dimensional structure of the insulin receptor kinase domain, which is 84% homologous to the IGF-1R kinase domain. The most potent inhibitor found in these assays was tyrphostin AG 538, with an IC(50) = 400 nM. In computer modeling, AG 538 was placed in the kinase domain of the insulin receptor and was able to sit in place of tyrosines 1158 and 1162, which undergo autophosphorylation. Experimentally it is indeed found that AG 538 does not compete with ATP but competes with the IGF-1R substrate. We prepared I-OMe AG 538, which is more hydrophobic and less sensitive to oxidation than AG 538. Both AG 538 and I-OMe AG 538 inhibit IGR-1R autophosphorylation in intact cells in a dose-dependent manner but I-OMe-AG 538 is superior, probably because of its enhanced hydrophobic nature. Both compounds inhibit the activation of the downstream targets PKB and Erk2. These findings suggest that AG 538 and I-OMe-AG 538 can serve as a lead compound for the development of substrate competitive inhibitors of the IGF-1R. The possible advantage of substrate competitive inhibitors vis-à-vis ATP competitive inhibitors is discussed.

3T3 Cells↗
PubMed2000

Inhibition of murine AIDS (MAIDS) development in C57BL/6J mice by tyrphostin AG-1387.

We previously showed that certain tyrphostin derivatives, known as protein tyrosine kinase inhibitors, also act as topoisomerase I-specific antagonists and inhibit Moloney murine leukemia virus replication in vitro in acutely and chronically infected cells. However, an accurate portrayal of retroviral-induced disease cannot rely exclusively on extrapolations from in vitro data. Therefore, experiments with animal models are essential for evaluating the efficacy of a specific drug in vivo. In this study, we examined the effect of tyrphostin AG-1387 on murine AIDS (MAIDS) development in C57BL/6J mice injected with the LP-BM5 virus mixture. A single dose of tyrphostin, administered together with or 24 h post virus inoculation, decreased the development of MAIDS symptoms as measured by spleen and lymph node weight, the T-cell response to concanavalin A (con A), and spleen architecture. Furthermore, weekly treatment with tyrphostins totally abolished MAIDS symptoms and prevented the viral infection of the spleen cells as measured by the absence of viral RNA and the restoration of T-cell function in these spleens. These results implicate that prolonged treatment with tyrphostins is needed for the prevention of MAIDS development in infected mice and suggest that it may be applied as a legitimate remedy for the treatment of retroviral-induced diseases.

Animals↗
PubMed2000

Deregulated expression of homeobox-containing genes, HOXB6, B8, C8, C9, and Cdx-1, in human colon cancer cell lines.

Previously we have demonstrated a reciprocal deregulation of various homeobox genes (HOXB6, B8, C8 and C9 vs Cdx-1) in human colorectal cancer (CRC). In the present study, using RT-PCR, we have investigated the expression pattern of these homeobox genes in various human colon cell lines, representing various stages of colon cancer progression and differentiation. Thus, we have tested polyposis coli Pc/AA adenoma cells, Caco-2, HT-29 and LS174T adenocarcinoma cell lines. All cell lines, except LS174T, demonstrated a pattern of deregulated homeobox gene expression which resembled that of CRC. In contrast, the pattern of expression of these genes in the highly oncogenic LS174T cells, as well as in Caco-2 cells transfected with activated Ha-ras or Polyoma middle T oncogene, resembled that of the normal mucosa. The reciprocal deregulation of HOX and Cdx-1 genes in CRC and in CRC-derived cell lines suggests a possible role in human CRC development.

Adenocarcinoma↗
PubMed2000

Disruption of microtubules in living cells by tyrphostin AG-1714.

Tyrphostin AG-1714 and several related molecules with the general structure of nitro-benzene malononitrile (BMN) disrupt microtubules in a large variety of cultured cells. This process can be inhibited by the stabilization of microtubules with taxol or by pretreatment of the cells with pervanadate, which inhibits tyrosine phosphatases and increases the overall levels of phosphotyrosine in cells. Unlike other microtubule-disrupting drugs such as nocodazole or colchicine, tyrphostin AG-1714 does not interfere with microtubule polymerization or stability in vitro, suggesting that the effect of this tyrphostin on microtubules is indirect. These results imply an involvement of protein tyrosine phosphorylation in the regulation of overall microtubule dynamics. Tyrphostins of AG-1714 type could thus be powerful tools for the identification of such microtubule regulatory pathways.

Animals↗
PubMed2000

Suboptimal splice sites of equine infectious anaemia virus control Rev responsiveness.

The Rev protein of equine infectious anaemia virus (EIAV) was shown previously to stimulate the expression of a heterologous CAT reporter gene when the 3' half of the EIAV genome was present downstream in cis. However, computer analysis could not reveal the existence of a stable RNA secondary structure that could be analogous to the Rev-responsive element of other lentiviruses. In the present study, the inhibitory RNA element designated the cis-acting repressing sequence (CRS) has been localized to the centre of the EIAV genome. The inhibition exerted by this element could be overcome by supplying Rev in trans. The ability of the EIAV CRS to function in a heterologous context suggests that it does not require interactions with other viral proteins. Site-directed mutagenesis showed that the various centrally located suboptimal splice sites of the EIAV genome function as CRS and confer Rev-dependence on the CRS-containing transcripts. In addition, the data suggest that in canine Cf2Th cells, which are highly permissive for EIAV replication, CRS prevents nuclear export of CRS-containing transcripts and the supply of Rev relieves this suppression.

Animals↗
PubMed2000

Local delivery of platelet-derived growth factor receptor-specific tyrphostin inhibits neointimal formation in rats.

Signal transduction through the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) system is involved in the process of postangioplasty restenosis. Tyrphostins are low molecular weight inhibitors of protein tyrosine kinases. We assessed the antiproliferative effects of PDGFRbeta-specific tyrphostin AG-1295 in vitro and in vivo. AG-1295 significantly inhibited rat smooth muscle cell growth stimulated by PDGF-BB or FCS. This antiproliferative effect was paralleled by reversible reduction of the total phosphotyrosine level and the degree of PDGFRbeta phosphorylation by the drug in vitro. Local sustained delivery of the drug from perivascularly implanted polymeric matrices resulted in focal AG-1295 levels of 711 and 29.1 ng/mg of dry arterial tissue 1 and 14 days after implantation in rats. AG-1295 delivered from polymeric matrices resulted in a 35% reduction of neointimal formation on day 14 after balloon injury in the rat carotid model. Tyrosine phosphorylation of certain transduction proteins in arterial tissue extracts was significantly upregulated by balloon injury on day 3 but was essentially returned to or below basal levels 14 days after injury. Tyrphostin treatment decreased tyrosine phosphorylation at both time points below the basal levels. Moreover, the enhancement of PDGFRbeta expression 3 and 14 days after arterial injury was strongly inhibited by AG-1295 treatment. It can be concluded that AG-1295 reduces neointimal formation by inhibiting PDGFbeta-triggered tyrosine phosphorylation.

Angioplasty, Balloon↗
PubMed1999

Human frizzled 1 interacts with transforming Wnts to transduce a TCF dependent transcriptional response.

The human homologue of fz1 (Hfz1) was cloned from a cDNA library. Hfz1 was shown to couple to Wnt signal transduction pathways by its ability to enhance Wnt induced TCF dependent transcription in both autocrine and paracrine modes. Enhanced TCF dependent signaling was dose dependent with respect to both Wnt-3A and Hfz1. Moreover, Hfz1 deletion mutants with truncated carboxy termini showed markedly reduced capacity to enhance Wnt signal transduction. Specificity was demonstrated with respect to signal transduction by different Wnts. While Wnt-3a, -3, -1 and to a lesser extent Wnt-2 cooperated with Hfz1 in the paracrine assay for TCF dependent signaling, neither Wnt-4, -5a, -5b, -6, -7a nor -7b did so, despite similar levels of expression. However, coimmunoprecipitation of Hfz1 with both Wnt-3a and Wnt-5a indicated that TCF dependent signaling in response to Wnts is not determined solely by their ability to bind the receptor. All of these findings provide strong evidence that Hfz1 is a functional partner for certain Wnts in inducing TCF dependent transcription.

Cell Line↗
PubMed1999

A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling.

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.

3T3 Cells↗
PubMed1999

Interaction of frizzled related protein (FRP) with Wnt ligands and the frizzled receptor suggests alternative mechanisms for FRP inhibition of Wnt signaling.

Frizzled related proteins (FRPs) comprise a family of secreted molecules that contain an N-terminal cysteine-rich domain (CRD) highly similar to the CRDs of the frizzled family of membrane-anchored Wnt receptors. FRPs have been shown to interact with Wnt proteins and antagonize Wnt signaling in a Xenopus developmental model. We demonstrated that FRP antagonizes the Wnt-induced increase in uncomplexed beta-catenin in both transient cotransfection and stable transformation models, where Wnt-induced morphological alterations are inhibited as well. We showed further that FRP inhibits Wnt signaling in a paracrine mode using a T-cell factor luciferase reporter to measure Wnt function. Investigation of the mechanisms responsible for FRP inhibition revealed that FRP forms complexes with WNT-1 or WNT-2 through its CRD domain. Transfection analysis with FRPs containing different tags revealed that FRP itself forms complexes and that this ability is conferred by its CRD domain. Finally, we demonstrated by cotransfection that FRP forms complexes with a prototype frizzled. All of these findings are consistent with a model by which FRP inhibits Wnt signaling through interactions with Wnt and/or formation of nonfunctional complexes with the frizzled receptor.

Animals↗
PubMed1999

Effects of the tyrosine kinase inhibitor AG957 and an Anti-Fas receptor antibody on CD34(+) chronic myelogenous leukemia progenitor cells.

The hallmark of chronic myelogenous leukemia (CML) is the Philadelphia (Ph) chromosome that fuses genetic sequences of the BCR gene on chromosome 22 with c-ABL sequences translocated from chromosome 9. BCR/ABL fusion proteins have a dysregulated protein tyrosine kinase (PTK) activity exerting a key role in malignant transformation. Targeting the tyrosine kinase activity of BCR/ABL or using agents capable of triggering apoptosis might represent attractive therapeutic approaches for ex vivo purging. AG957, a member of the tyrphostin compounds, exerts a selective inhibition of p210(BCR/ABL) tyrosine phosphorylation. We report here that preincubation of CML or normal CD34(+) cells with graded concentration of AG957 (1 to 100 micromol/L) resulted in a statistically significant, dose-dependent suppression of colony growth from multipotent, erythroid, and granulocyte-macrophage progenitors as well as the more primitive long-term culture-initiating cells (LTC-IC). However, AG957 doses causing 50% inhibition (ID50) of CML and normal progenitors were significantly different for multilineage colony-forming units (CFU-Mix; 12 v 64 micromol/L; P =.008), burst-forming unit-erythroid (BFU-E; 29 v 89 micromol/L; P =.004), colony-forming unit-granulocyte-macrophage (CFU-GM; 34 v 85 micromol/L; P =.004), and LTC-IC (43 v 181 micromol/L; P =.004). In 5 of 10 patients, analysis of BCR/ABL mRNA on single progenitors by reverse transcription-polymerase chain reaction showed that AG957 at 50 micromol/L significantly reduced the mean (+/-SD) percentage of BCR/ABL-positive progenitors (92% +/- 10% v 33 +/- 5%; P =.001). Because AG957 treatment resulted in significantly higher percentages of apoptotic cells (30% v 9%) in the BCR/ABL-transfected 32DLG7 cells as compared with 32D-T2/93 cells (BCR/ABL-negative), we investigated the combined effects of AG957 with the anti-Fas receptor (Fas-R) monoclonal antibody CH11 that triggers apoptosis. As compared with AG957 alone, the sequential treatment of CML CD34(+) cells with AG957 (1 micromol/L) and CH11 (1 microgram/mL) increased CFU-Mix, BFU-E, and CFU-GM growth inhibition by 1.6-fold, 3-fold, and 4-fold, respectively. In contrast, the treatment of normal CD34(+) cells with AG957 and CH11 failed to enhance AG957-induced colony growth inhibition. We conclude that (1) AG957 inhibits in a dose-dependent manner CML CD34-derived colony formation by both primitive LTC-IC as well as committed CFU-Mix, BFU-E, and CFU-GM; (2) this growth inhibition is associated with the selection of a substantial amount of BCR/ABL-negative progenitors; and (3) the antiproliferative effect of AG957 is dramatically increased by combining this compound with the anti-Fas-R antibody CH11. These data may have significant therapeutic applications.

Adult↗
PubMed1999

Alpha-cyanocinnamide derivatives: a new family of non-peptide, non-sulfhydryl inhibitors of Ras farnesylation.

Farnesylation of Ras and other proteins is required for their membrane attachment and normal function. Here we report on the synthesis of alpha-cyanocinnamide derivatives, a new family of farnesyltransferase inhibitors. These compounds are nonpeptidic and do not contain sulfhydryl groups. The most potent compound is a pure competitive inhibitor with respect to the Ras protein and mixed competitive with respect to farnesyl diphosphate. Selectivity studies against geranylgeranyltransferase and biological activities of selected compounds are described.

3T3 Cells↗
PubMed1999

Crystal structure of Hck in complex with a Src family-selective tyrosine kinase inhibitor.

The crystal structure of the autoinhibited form of Hck has been determined at 2.0 A resolution, in complex with a specific pyrazolo pyrimidine-type inhibitor, PP1. The activation segment, a key regulatory component of the catalytic domain, is unphosphorylated and is visualized in its entirety. Tyr-416, the site of activating autophosphorylation in the Src family kinases, is positioned such that access to the catalytic machinery is blocked. PP1 is bound at the ATP-binding site of the kinase, and a methylphenyl group on PP1 is inserted into an adjacent hydrophobic pocket. The enlargement of this pocket in autoinhibited Src kinases suggests a route toward the development of inhibitors that are specific for the inactive forms of these proteins.

Animals↗
PubMed1999

Growth inhibition of psoriatic keratinocytes by quinazoline tyrosine kinase inhibitors.

Psoriasis is characterized by hyperproliferation of keratinocytes associated with an inflammatory infiltrate in the epidermis. Among factors which may be related to hyperplasia of psoriatic keratinocytes is the persistent autocrine stimulation of the epidermal growth factor receptor (EGFR) by transforming growth factor-alpha. Owing to the pivotal role of the EGFR in driving the growth of human psoriatic keratinocytes, we examined two selective inhibitors of EGFR kinase activity: 4-(3-bromophenylamino)-6, 7-dimethoxyquinazoline (AG1517/SU5271) and 4-(3-chlorophenylamino)-6, 7-dimethoxyquinazoline (AG1478) on psoriatic keratinocytes. SU5271 potently inhibits ligand-induced autophosphorylation of EGFR, and downstream signal transduction events, including DNA replication and cell cycle progression. SU5271, at micromolar concentrations, inhibited the proliferation of keratinocytes isolated from psoriatic lesions in excellent correlation with its EGFR kinase inhibitory activity in these cells. Biologically active concentrations of SU5271 penetrated human cadaver skin, suggesting that this compound is a strong candidate as an antipsoriatic agent.

Animals↗