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Biomedical subjects

D Ilić

Publications and source records attributed to D Ilić.

At least 19 recordsLinked to original sources
PubMed2002

Biological and clinical significance of clonogenic assays in patients with myelodysplastic syndromes.

Biological and clinical significance of growth pattern of hematopoietic progenitors were investigated in 117 patients with primary myelodysplastic syndromes (MDSs) at referral. Abnormal (i.e., "leukemic" or absent) growth of GM colonies (CFU-GM) and GM clusters was found in 47% of patients with "advanced" MDS (RAEB, RAEB-t, and CMML) and in 15% of "low-risk" (RA/RARS) patients. In vitro erythropoiesis was decreased in most of the patients, with significantly lower number of BFU-E in "advanced" MDS than in RA/RARS patients. Megakaryocyte progenitors (CFU-MK) were very low or absent in almost all the patients, regardless of the FAB type. Significant correlation was demonstrated between the number of BFU-E and hemoglobin concentration and between number of CFU-MK and platelet count. Growth capacity of GM progenitors appears to be in proportion to "myeloproliferative" capacity of the malignant clone. T-cell depletion had no influence on growth capacity of hematopoietic progenitors, nor did colony growth respond in a dose-dependent manner to different concentrations of LCM. Growth capacity of MDS hematopoietic progenitors was independent of Bournemouth score, of the presence and type of cytogenetic abnormality, and of the expression of CD95 and caspase-3 antigens on bone marrow cells. However, in patients with "abnormal" growth of GM progenitors, CD34 antigen expression was significantly higher than in patients with "normal" growth. "Abnormal" GM growth was found to be independently predictive regarding the survival and the risk for AML development. In contrast, the prognostic value of erythroid and megakaryocyte cultures was found to be limited.

Adolescent↗
PubMed2001

Plasma membrane-associated pY397FAK is a marker of cytotrophoblast invasion in vivo and in vitro.

During human pregnancy specialized placental cells of fetal origin, termed cytotrophoblasts, invade the uterus and its blood vessels. This tumor-like process anchors the conceptus to the mother and diverts the flow of uterine blood to the placenta. Previously, we showed that the expression of molecules with important functional roles, including a number of extracellular matrix integrin receptors, is precisely modulated during cytotrophoblast invasion in situ. Here we exploited this observation to study the role of the focal adhesion kinase (FAK), which transduces signals from the extracellular matrix and recruits additional signaling proteins to focal adhesions. Immunolocalization studies on tissue sections showed that FAK is expressed by cytotrophoblasts in all stages of differentiation. Because extracellular matrix-induced integrin clustering results in FAK (auto)phosphorylation on tyrosine 397 (Y397FAK), we also localized this form of the molecule. Immunolocalization experiments detected Y397FAK in a subset of cytotrophoblasts near the surface of the uterine wall. To assess the functional relevance of this observation, we used an adenovirus strategy to inhibit cytotrophoblast expression of FAK as the cells differentiated along the invasive pathway in vitro. Compared to control cells transduced with a wild-type virus, cytotrophoblasts that expressed antisense FAK exhibited a striking reduction in their ability to invade an extracellular matrix substrate. When cytotrophoblast differentiation was compromised (hypoxia in vitro, preeclampsia in vivo), Y397FAK levels associated with the plasma membrane were strikingly lower, although total FAK levels did not change. Together our results suggest that (auto)phosphorylation of Y397 on FAK is a critical component of the signaling pathway that mediates cytotrophoblast migration/invasion.

Biomarkers↗
PubMed2001

Focal adhesion kinase and p130Cas mediate both sarcomeric organization and activation of genes associated with cardiac myocyte hypertrophy.

Hypertrophic terminally differentiated cardiac myocytes show increased sarcomeric organization and altered gene expression. Previously, we established a role for the nonreceptor tyrosine kinase Src in signaling cardiac myocyte hypertrophy. Here we report evidence that p130Cas (Cas) and focal adhesion kinase (FAK) regulate this process. In neonatal cardiac myocytes, tyrosine phosphorylation of Cas and FAK increased upon endothelin (ET) stimulation. FAK, Cas, and paxillin were localized in sarcomeric Z-lines, suggesting that the Z-line is an important signaling locus in these cells. Cas, alone or in cooperation with Src, modulated basal and ET-stimulated atrial natriuretic peptide (ANP) gene promoter activity, a marker of cardiac hypertrophy. Expression of the C-terminal focal adhesion-targeting domain of FAK interfered with localization of endogenous FAK to Z-lines. Expression of the Cas-binding proline-rich region 1 of FAK hindered association of Cas with FAK and impaired the structural stability of sarcomeres. Collectively, these results suggest that interaction of Cas with FAK, together with their localization to Z-lines, is critical to assembly of sarcomeric units in cardiac myocytes in culture. Moreover, expression of the focal adhesion-targeting and/or the Cas-binding proline-rich regions of FAK inhibited ANP promoter activity and suppressed ET-induced ANP and brain natriuretic peptide gene expression. In summary, assembly of signaling complexes that include the focal adhesion proteins Cas, FAK, and paxillin at Z-lines in the cardiac myocyte may regulate, either directly or indirectly, both cytoskeletal organization and gene expression associated with cardiac myocyte hypertrophy.

Animals↗
PubMed2001

Integrin alphavbeta3 mediates K1735 murine melanoma cell motility in vivo and in vitro.

The integrin alphavbeta3 has been shown to be tightly linked to progression of human melanoma. In this study, using two clones from the K1735 murine melanoma system, we investigated the role of alphavbeta3 in metastasis. The highly metastatic K1735M2 cells express the alphavbeta3 integrin, whereas the poorly metastatic K1735C23 cells do not. When transduced with the beta3 integrin subunit cDNA, the K1735C23 cells produced lung lesions and, in two animals, cardiac metastases, whereas the parental C23 cells did not. By contrast, transduction of the full-length beta3 integrin antisense DNA into the K1735M2 cells suppressed metastatic colonization. To specifically investigate the activation of beta3 integrin-mediated pathways, the beta3-positive and the beta3-negative K1735 cells were plated onto vitronectin, a major matrix molecule of both primary and metastatic melanomas. Tyr397 of FAK was phosphorylated several times higher in beta3-expressing K1735 melanoma cells than in beta3-negative cells. To determine whether phosphorylation of FAK was associated with K1735 melanoma motility, we expressed the FAK-related non-kinase (FRNK) in the highly metastatic K1735M2 cells. Exogenous expression of FRNK suppressed phosphorylation of FAK at Tyr397 and decreased the invasive ability of these cells. In addition, expression of a constitutively active mutant Src in poorly metastatic K1735C23 cells increased invasion in vitro; whereas expression of a kinase-inactive Src mutant suppressed invasion. Our results suggest that signals initiated by alphavbeta3 promote metastasis in K1735 melanoma cells through the phosphorylation of FAK and activation of Src.

Animals↗
PubMed2000

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 (Ilić 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.

Animals↗
PubMed1998

c-Src activation plays a role in endothelin-dependent hypertrophy of the cardiac myocyte.

Activation of the atrial natriuretic peptide (ANP) gene is regarded as one of the earliest and most reliable markers of hypertrophy in the ventricular cardiac myocyte. We have examined the role of the nonreceptor tyrosine kinases in the signaling mechanism(s) leading to hypertrophy using human ANP gene promoter activity as a marker. Endothelin (ET), a well known hypertrophic agonist, increased activity of c-Src, c-Yes, and Fyn within minutes and promoted a selective redistribution of each of these kinases within the cell. Overexpression of c-Src effected a significant increase in activity of a cotransfected human ANP promoter-driven chloramphenicol acetyl transferase reporter, while expression of either c-Yes or Fyn was considerably less effective in this regard. ET-dependent stimulation of the human ANP gene promoter was partially inhibited by co-transfection with dominant negative Ras or dominant negative Src or Csk or by treatment with the potent Src family-selective tyrosine kinase inhibitor PP1, suggesting that the Src family kinases are involved in signaling ET-dependent activation of this promoter. Both ET- and Src-dependent activation of the ANP promoter required the presence of a CArG motif in a serum response element-like structure between -422 and -413 but did not appear to require assembly of a ternary complex for full activity. These findings support a role for Src in the activation of ANP gene expression and suggest that this kinase may contribute in an important way to the signaling mechanisms that activate hypertrophy in the cardiac myocyte.

Animals↗
PubMed1998

Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis.

In many malignant cells, both the anchorage requirement for survival and the function of the p53 tumor suppressor gene are subverted. These effects are consistent with the hypothesis that survival signals from extracellular matrix (ECM) suppress a p53-regulated cell death pathway. We report that survival signals from fibronectin are transduced by the focal adhesion kinase (FAK). If FAK or the correct ECM is absent, cells enter apoptosis through a p53-dependent pathway activated by protein kinase C lambda/iota and cytosolic phospholipase A2. This pathway is suppressible by dominant-negative p53 and Bcl2 but not CrmA. Upon inactivation of p53, cells survive even if they lack matrix signals or FAK. This is the first report that p53 monitors survival signals from ECM/FAK in anchorage- dependent cells.

Animals↗
PubMed1998

Pyk2 and Src-family protein-tyrosine kinases compensate for the loss of FAK in fibronectin-stimulated signaling events but Pyk2 does not fully function to enhance FAK- cell migration.

The focal adhesion kinase (FAK) protein-tyrosine kinase (PTK) links transmembrane integrin receptors to intracellular signaling pathways. We show that expression of the FAK-related PTK, Pyk2, is elevated in fibroblasts isolated from murine fak-/- embryos (FAK-) compared with cells from fak+/+ embryos (FAK+). Pyk2 was localized to perinuclear regions in both FAK+ and FAK- cells. Pyk2 tyrosine phosphorylation was enhanced by fibronectin (FN) stimulation of FAK- but not FAK+ cells. Increased Pyk2 tyrosine phosphorylation paralleled the time-course of Grb2 binding to Shc and activation of ERK2 in FAK- cells. Pyk2 in vitro autophosphorylation activity was not enhanced by FN plating of FAK- cells. However, Pyk2 associated with active Src-family PTKs after FN but not poly-L-lysine replating of the FAK- cells. Overexpression of both wild-type (WT) and kinase-inactive (Ala457), but not the autophosphorylation site mutant (Phe402) Pyk2, enhanced endogenous FN-stimulated c-Src in vitro kinase activity in FAK- cells, but only WT Pyk2 overexpression enhanced FN-stimulated activation of co-transfected ERK2. Interestingly, Pyk2 overexpression only weakly augmented FAK- cell migration to FN whereas transient FAK expression promoted FAK- cell migration to FN efficiently compared with FAK+ cells. Significantly, repression of endogenous Src-family PTK activity by p50(csk) overexpression inhibited FN-stimulated cell spreading, Pyk2 tyrosine phosphorylation, Grb2 binding to Shc, and ERK2 activation in the FAK- but not in FAK+ cells. These studies show that Pyk2 and Src-family PTKs combine to promote FN-stimulated signaling events to ERK2 in the absence of FAK, but that these signaling events are not sufficient to overcome the FAK- cell migration defects.

Amino Acid Sequence↗
PubMed1997

Skin abnormality in aged fyn-/- fak+/- mice.

Focal adhesion kinase (FAK) is a novel non-receptor protein tyrosine kinase implicated in transducing signals from cell surface receptors. Its association with Fyn, a member of the Src family of tyrosine kinases, has been observed in cell lines. To examine in vivo the interaction between these two proteins, Fyn-deficient mice were bred with fak heterozygous mutants (Fak deficiency is embryonic lethal). A majority of animals with the double mutation (fyn-/- fak+/-) displayed a transient impairment in thymocyte development at four weeks of age. However, all of them developed skin abnormalities at the age of 8-12 months. The most prominent among abnormalities was a greatly increased number and size of sebaceous glands. Also, the epidermis was thickened and hyperkeratotic. These observations would suggest involvement of Fyn and FAK in keratinocyte differentiation.

Age Factors↗
PubMed1997

Focal adhesion kinase: at the crossroads of signal transduction.

Morphogenetic processes during development, including cell migration, depend on signals from both the extracellular matrix (ECM) and soluble signaling factors. Extensive evidence has shown that the nonreceptor tyrosine kinase, focal adhesion kinase (FAK), is activated in response to both kind of signal. The most definitive evidence that FAK is directly downstream of signals initiated by the ECM comes from comparing the phenotypes of mice deficient for FAK and the ECM molecule, fibronectin: in both cases embryos die at about E8.5 and display almost identical severe vascular and other mesodermal defects. It is now clear that there are additional FAK-like proteins, indicating the existence of a FAK family. Furthermore, FAK is not located at adhesive sites in all cells where it is expressed. This, plus extensive data indicating that FAK becomes activated in response to several soluble signaling factors, suggests that the FAK family may be at the crossroads of multiple signaling pathways that affect cell and developmental processes.

Animals↗
PubMed1996

Impairment of mobility in endodermal cells by FAK deficiency.

Focal adhesion kinase (FAK) is a novel nonreceptor protein tyrosine kinase that localizes in focal adhesions. It is expressed in a variety of cell types, and we reported earlier that is deficiency causes a decrease of mobility in mesodermal cells with enhanced formation of focal adhesions. With embryoid bodies generated from embryonic stem cells, we also observed a decrease of mobility in FAK-deficient endodermal cells with enhanced focal adhesion formation.

Animals↗
PubMed1996

p59fyn-p125FAK cooperation in development of CD4+CD8+ thymocytes.

p59fyn is an Src family nonreceptor tyrosine kinase that has been suggested to play an important role in T-cell development and function. p125FAK is a unique nonreceptor tyrosine kinase and has been known to respond to integrin-extracellular matrix interactions. To examine their roles in thymocytes, heterozygous fak mutation was introduced into homozygous Fyn deficiency. The double mutation, but neither Fyn deficiency nor FAK heterozygosity alone, displayed impaired development of CD4+CD8+ thymocytes with atrophy of the thymic cortex, suggesting a unique cooperation between p59fyn and p125FAK in CD4+CD8+ T-cell development.

Animals↗
PubMed1995

Mesodermal defect in late phase of gastrulation by a targeted mutation of focal adhesion kinase, FAK.

FAK is a unique non-receptor protein tyrosine kinase that was found in cellular focal adhesions. An increasing number of in vitro observations has suggested that FAK mediates signaling through integrins brought about by interactions with extracellular matrix (ECM). It is highly tyrosine-phosphorylated in v-src-transformed cells and during embryogenesis. To clarify the function of FAK in cell-ECM interactions, embryonic phenotype of its mutant was analysed. FAK-deficient embryos could implant and initiate gastrulation normally, but showed abnormalities in subsequent development. The abnormalities were characterized as a general deficiency in mesoderm, and the phenotype was quite similar to that caused by fibronectin-deficiency. The results suggest that FAK mediates fibronectin-integrin interactions uniquely at this stage of development, thereby playing an essential role in development of mesodermal cell lineages.

Animals↗
PubMed1995

Reduced cell motility and enhanced focal adhesion contact formation in cells from FAK-deficient mice.

The intracellular protein tyrosine kinase FAK (focal adhesion kinase) was originally identified gy its high level of tyrosine phosphorylation in v-src-transformed cells. FAK is also highly phosphorylated during early development. In cultured cells it is localized to focal adhesion contacts and becomes phosphorylated and activated in response to integrin-mediated binding of cells to the extracellular matrix, suggesting an important role in cell adhesion and/or migration. We have generated FAK-deficient mice by gene targeting to examine the role of FAK during development. Mutant embryos displayed a general defect of mesoderm development, and cells from these embryos had reduced mobility in vitro. Surprisingly, the number of focal adhesions was increased in FAK-deficient cells, suggesting that FAK may be involved in the turnover of focal adhesion contacts during cell migration.

Animals↗
PubMed1990

Detection of cross-reacting idiotypes in sera of lymphoma patients by inverse monoclonal radioimmunoassay.

An anti-idiotypic IgG1 kappa murine monoclonal antibody (MoAb) Y7 against purified monoclonal IgM lambda 1, derived from a patient with Waldenström's macroglobulinemia, has been generated. This antibody cross-reacted with the tumor-derived idiotypes of patients with B cell non Hodgkin's lymphoma as measured by competitive inverse solid radioimmunoassay using unpurified serum samples. Our results with the inhibition curves of 10 sera of normal donors and 60 sera of lymphoma patients indicate that 21 lymphoma patients revealed cross-reactivities greater than 7%, the mean value observed in normal donors. Of these, 5 sera cross-reacted strongly, in the range of 43-163%, revealing a frequency of positive cross-reactivity for MoAb Y7 of 1/12 sera of lymphoma patients. The generation of a panel of anti-idiotypic antibodies which cross-react with different tumor-derived Ig in serum may be valuable for monitoring the disease in a high proportion of NHL patients.

Antibodies, Monoclonal↗
PubMed1990

[Characteristics of the ventilatory status of the lungs in workers in the electronics industry].

To establish the effects of occupational respiratory noxae on the state of the bronchopulmonary system a study of the working environment and examinations of workers from the electronics industry whose workplaces were near tin baths and in conductor soldering shops were carried out. The following chemical noxae were found to be present in the working environment: lead, tin, calophonium and ethyl alcohol, all in permitted concentrations. Comparison of the state of lung ventilation in workers from the electronics industry (n = 120) and control workers (n = 129) pointed to a major increase in total air flow resistance during calm breathing in the exposed workers. The obstructive changes were mainly localized in the small airways.

Adult↗