FAK Gene Research Articles

Abstract 2635: Galectin-1 modulates glioblastoma cell migration via interaction with FAK.

Abstract Malignant glioblastomas (GBM) are the most common brain tumors and exhibit excessive growth, cell cycle regulation, neovascularization, angiogenesis, migration, immune escape and resistance to apoptosis. Galectin-1 (Gal-1) is a 14.5-kDa β-galactoside-binding protein, which displays intracellular (i.e., protein-protein interactions) and extracellular (i.e., protein-oligosaccharide interactions) functions with marked pro-angiogenic and pro-migratory effects in gliomas. Although glioma is considered one of the deadliest cancers with median survival ranging from nine to twelve months, therapeutic treatments that target Gal-1 may prove effective since Gal-1 is one of the genes that cause aggressive behaviors of human GBM such as migration and invasion. Gal-1 controls glioma cell migration and invasion by modifying the actin cytoskeleton and expression of small GTPases. In the present study, immunohistochemical, Real Time PCR and Western blot experiments performed with clinical samples demonstrated that Gal-1 is highly expressed in human GBM tissue as compared to healthy brain tissue. Matrigel invasion assays confirmed that treatment of glioma cell lines U251 and 5310 with human umbilical cord blood stem cells (hUCBSC) almost completely blocked invasion of GBM cells. We also observed that treatment of GBM cells with hUCBSC decreased the expression of both Gal-1 and FAK. Additionally, silencing either the Gal-1 or FAK gene using shRNA decreased migration and invasion of glioblastoma cells. Furthermore, using immunocytochemistry and Western blot analyses, we observed that Gal-1 and FAK interact with each other in glioblastoma. Taken together, our results suggest that Gal-1 and FAK might have a direct protein-protein interaction for their function in the tumorigenesis of glioblastoma. Citation Format: Kiran Kumar Velpula, Arnima Bhasin, Jane S. Zhang, Andrew J. Tsung, Krishna Kumar Veeravalli, Jasti S. Rao, Venkata Ramesh Dasari. Galectin-1 modulates glioblastoma cell migration via interaction with FAK. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2635. doi:10.1158/1538-7445.AM2013-2635

Focal Adhesion Kinase Inactivation Reduces the Development of Acute Leukemia and Partially Rescues Hematopoietic Stem Cell Defects in Pten-Knockout Mice

AbstractAbstract 864Phosphatase and tensin homolog on chromosome 10 (Pten) is a tumor suppressor which possesses both lipid and protein phosphatase activities. Mutations and epigenetic inactivations of the Pten gene are commonly detected in a large number of tissue malignancies, including leukemias and lymphomas. Studies using Hematopoietic Pten-knockout in adult mice (Pten−/−) have demonstrated that Pten plays a critical role in maintaining the homeostasis of bone marrow (BM) hematopoiesis. Pten inactivation promotes the proliferation and peripheral mobilization of BM hematopoietic stem cells (HSCs). Pten−/− mice develop myeloproliferative disorders (MPD) within days, followed by acute leukemic transformation. Most previous studies attributed such phenotypic changes observed in Pten−/− mice to excessive activation of the PI3K/AKT/mTOR signal, a consequence of the loss of Pten’s lipid phosphatase activity. However, the role of Pten’s protein phosphatase activity in the regulation of HSCs and leukemogenesis is not well studied.Focal adhesion kinase (Fak) is a critical substrate for the protein phosphatase activity of Pten. Dysregulation of Fak has been observed in many cancers, including acute myeloid leukemias (AML) and acute lymphocytic leukemias (ALL). Therefore, we postulated that Fak might play a pivotal role in the development and progression of leukemia following Pten deletion. To test this hypothesis, we generated Mx1-Cre+Ptenfl/flFakfl/fl mice (an interferon-inducible Pten and Fak compound-knockout, Pten−/−Fak−/−) in which both the Pten and Fak genes in the hematopoietic system are deleted upon injection of polyinosinic-polycytidylic acid (pI-pC). Our results showed that the genetic inactivation of Fak can partially rescue HSC defects associated with Pten deficiency. We found that peripheral mobilization of HSCs in Pten−/−Fak−/− mice is significantly reduced compared to Pten−/− mice. As a consequence, more long-term HSCs (LT-HSCs) are preserved in the BM of Pten−/−Fak−/− mice compared to Pten−/− mice. Transplantation studies suggested that the hematopoietic reconstitutive capacity of Pten−/−Fak−/− HSCs is significantly improved compared to Pten−/− HSCs. Although Fak deletion fails to prevent the development of MPD in Pten−/− mice, Fak deletion does significantly reduce the frequency of AML/ALL, also significantly delays the onset of AML/ALL in comparison to Pten−/− mice. This study suggests that Fak might be a potential target for preventing the MPD-to-AML/ALL transformation and therefore blocking the Fak activity may hold a promise for a novel anti-leukemia therapy. The molecular mechanisms underlying the phenotype restoration of Pten−/− mice by Fak deletion in the hematopoietic system are actively being studied in our laboratory. Disclosures:No relevant conflicts of interest to declare.

Effect of FAK, DLC-1 gene expression on OVCAR-3 proliferation

The study investigates the effect of FAK, DLC-1 on OVCAR-3 proliferation. FAK gene siRNA vector recombinant plasmid was constructed using RNA interference technique. FAK gene-transfected OVCAR-3 cells, OVCAR-3 cells with DLC-1 gene expression, and OVCAR-3 cells with simultaneous expression of DLC-1 and FAK genes were obtained using gene transfection technology. In addition, siRNA control group and blank control were also given. Effect of FAK, DLC-1 gene expression on OVCAR-3 proliferation was examined by FCM and Cell Counting Kit-8 (CCK-8) methods. Results showed that DLC-1 gene high expression and FAK gene silencing, single silencing FAK gene, and single DLC-1 gene high expression in OVCAR-3 cells may decrease S and G2/M phase proportion of the cell cycle. Moreover, DLC-1 gene high expression and FAK gene silencing in OVCAR-3 cells can display the most significant effect. This confirmed that DLC-1 gene high expression and FAK gene silencing may significantly inhibit the OVCAR-3 cells proliferation. CCK-8 analysis showed that silence FAK gene expression or/and increasing DLC-1 gene expression may decrease OVCAR-3 growth rate. Moreover, simultaneous silence the expression of FAK gene and high expression of DLC-1 gene can display the most significant effect on OVCAR-3 growth. It can be concluded that downregulation of FAK gene expression or/and upregulation of DLC-1 gene expression can all inhibit the OVCAR-3 growth. Moreover, DLC-1 gene expression and FAK gene silencing can display the most marked inhibitory effect on the OVCAR-3 growth.

Activation of Endogenous FAK via Expression of Its Amino Terminal Domain in Xenopus Embryos

BackgroundThe Focal Adhesion Kinase is a well studied tyrosine kinase involved in a wide number of cellular processes including cell adhesion and migration. It has also been shown to play important roles during embryonic development and targeted disruption of the FAK gene in mice results in embryonic lethality by day 8.5.Principal FindingsHere we examined the pattern of phosphorylation of FAK during Xenopus development and found that FAK is phosphorylated on all major tyrosine residues examined from early blastula stages well before any morphogenetic movements take place. We go on to show that FRNK fails to act as a dominant negative in the context of the early embryo and that the FERM domain has a major role in determining FAK’s localization at the plasma membrane. Finally, we show that autonomous expression of the FERM domain leads to the activation of endogenous FAK in a tyrosine 397 dependent fashion.ConclusionsOverall, our data suggest an important role for the FERM domain in the activation of FAK and indicate that integrin signalling plays a limited role in the in vivo activation of FAK at least during the early stages of development.

Effect of small interfering RNA transfection on FAK and DLC1 mRNA expression in OVCAR-3

RNA interference is an evolutionarily conserved cellular defense mechanism that protects cells from hostile genes and regulates the function of normal genes during growth and development. In this study, we established GFP-siFAK-DLC1 vector and transfect the vector into OVCAR-3 cells. RT-PCR and western blot analyses were performed for FAK, DLC1 mRNA, and protein expression in OVCAR-3 cells. ELISA method was used for caspase-3 and caspase-9 activities. These studies demonstrate that both recombinant pGFP-siFAK-DLC1 vector and pGFP-siCon-DLC1 vector may effectively promote DLC1 mRNA transcription and didn't affect siRNA effect. Recombinant vector (pGFP-siFAK-DLC1) may promote DLC1 gene expression, and effectively silence FAK gene expression. Silencing FAK mRNA expression and DLC1 mRNA expression may markedly enhance caspase-3 and caspase-9 activities in OVCAR-3 cells. These results showed that in ovarian cancer OVCAR-3 cell silencing FAK gene expression or / and increasing DLC-1 gene expression, could improve Caspase-3 and Caspase-9 protease activities. In the expression of DLC-1 and silence FAK expression group (double action group) effect was more significant as compared with the silence FAK gene group or expression of DLC-1 gene alone, difference was significant (p < 0.05).

An immediate-early protein of white spot syndrome virus modulates the phosphorylation of focal adhesion kinase of shrimp

WSSV interacts with integrin during infection of shrimps and modulate the focal adhesion kinase which is known as a regulator of several downstream signaling pathways. Viral protein kinases are thought to be important for virus infection by regulating the host signaling pathways. WSV083 is an immediate-early gene of white spot syndrome virus that contains a Ser/Thr protein kinase domain. So, does WSSV modulate FAK phosphorylation via the WSV083 molecule? In this study, co-transfection of WSV083 and MjFAK genes proceeded in insect cells revealed that the MjFAK phosphorylation and cell adhesion activity could be inhibited by the expression of WSV083. Kinase domain mutants of WSV083 lost its ability of inhibiting FAK phosphorylation. Moreover, silencing of FAK gene through RNAi accelerated the shrimp death rate upon WSSV challenge. These results demonstrate for the first time that modulation of FAK phosphorylation by WSV083 plays a critical role in the pathogenesis of WSSV infection.

The Effect of Conjugating RGD into 3D Alginate Hydrogels on Adipogenic Differentiation of Human Adipose‐Derived Stromal Cells

The effects of RGD peptide conjugation to alginate hydrogel on the adipogenic differentiation of ASCs was investigated. After 3 d of culture, RGD-modified alginate hydrogels significantly stimulated FAK and integrin α1 gene expressions and vinculin expression in ASCs. In addition, RGD-modified alginate hydrogels significantly enhanced the adipogenic differentiation of human ASCs to exhibit higher expression levels of oil red O staining and adipogenic genes compared to those of the control group (unmodified gels). These results suggest potential applications of RGD-modified alginate gels for adipose tissue regeneration.

Curcumin blocks migration and invasion of mouse-rat hybrid retina ganglion cells (N18) through the inhibition of MMP-2, -9, FAK, Rho A and Rock-1 gene expression.

Cancer metastasis involves multiple processes which may complicate clinical management and even lead to death. Matrix metalloproteinases (MMPs) play an important role in cancer cell invasion, metastasis and angiogenesis, depending on whether agents can inhibit MMPs which could lead to inhibition of the migration and invasion of cancer cells. Curcumin, the active constituent of the dietary spice turmeric, has potential for the prevention and therapy of cancer. However, there is no study to address the effects of curcumin on migration and invasion of mouse-rat hybrid retina ganglion cells (N18). This is the first study to explore the anti-migration and -invasion of curcumin in mouse-rat hybrid retina ganglion cells (N18) in vitro. Curcumin exerted a dose- and time-dependent inhibitory effect on the invasion and migration of N18 cells in vitro. Results from Western blotting showed that curcumin inhibited the protein levels of PKC, FAK, NF-kappaB p65 and Rho A leading to the inhibition of ERK1/2, MKK7, COX-2 and ROCK1, respectively, finally causing the inhibition of MMP-2 and -9 for the inhibition of migration and invasion of N18 cells. Moreover, this action was involved in the inhibition of gene expression of MMP-2 and -7, FAK, ROCK1 and Rho A. Overall, the above data show that the anticancer effect of curcumin also exists for the inhibition of migration and invasion in N18 cells, and that curcumin may be a powerful candidate for developing preventive agents for cancer metastasis.

Curcumin blocks migration and invasion of mouse-rat hybrid retina ganglion cells (N18) through the inhibition of MMP-2, -9, FAK, Rho A and Rock-1 gene expression

Cancer metastasis involves multiple processes which may complicate clinical management and even lead to death. Matrix metalloproteinases (MMPs) play an important role in cancer cell invasion, metastasis and angiogenesis, depending on whether agents can inhibit MMPs which could lead to inhibition of the migration and invasion of cancer cells. Curcumin, the active constituent of the dietary spice turmeric, has potential for the prevention and therapy of cancer. However, there is no study to address the effects of curcumin on migration and invasion of mouse-rat hybrid retina ganglion cells (N18). This is the first study to explore the anti-migration and -invasion of curcumin in mouse-rat hybrid retina ganglion cells (N18) in vitro. Curcumin exerted a dose- and time-dependent inhibitory effect on the invasion and migration of N18 cells in vitro. Results from Western blotting showed that curcumin inhibited the protein levels of PKC, FAK, NF-κB p65 and Rho A leading to the inhibition of ERK1/2, MKK7, COX-2 and ROCK1, respectively, finally causing the inhibition of MMP-2 and -9 for the inhibition of migration and invasion of N18 cells. Moreover, this action was involved in the inhibition of gene expression of MMP-2 and -7, FAK, ROCK1 and Rho A. Overall, the above data show that the anticancer effect of curcumin also exists for the inhibition of migration and invasion in N18 cells, and that curcumin may be a powerful candidate for developing preventive agents for cancer metastasis.

Focal Adhesion Kinase Signaling Regulates the Expression of Caveolin 3 and β1 Integrin, Genes Essential for Normal Myoblast Fusion

An essential phase of skeletal myogenesis is the fusion of mononucleated myoblasts to form multinucleated myotubes. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, but the mechanisms by which these proteins regulate cell fusion remain mostly unknown. Here, we focused on the role of focal adhesion kinase (FAK), an important nonreceptor protein tyrosine kinase involved in integrin signaling, as a potential mediator by which integrins may regulate myoblast fusion. To test this hypothesis in vivo, we generated mice in which the Fak gene was disrupted specifically in muscle stem cells ("satellite cells") and we found that this resulted in impaired myotube formation during muscle regeneration after injury. To examine the role of FAK in the fusion of myogenic cells, we examined the expression of FAK and the effects of FAK deletion on the differentiation of myoblasts in vitro. Differentiation of mouse primary myoblasts was accompanied by a rapid and transient increase of phosphorylated FAK. To investigate the requirement of FAK in myoblast fusion, we used two loss-of-function approaches (a dominant-negative inhibitor of FAK and FAK small interfering RNA [siRNA]). Inhibition of FAK resulted in markedly impaired fusion but did not inhibit other biochemical measures of myogenic differentiation, suggesting a specific role of FAK in the morphological changes of cell fusion as part of the differentiation program. To examine the mechanisms by which FAK may be regulating fusion, we used microarray analysis to identify the genes that failed to be normally regulated in cells that were fusion defective due to FAK inhibition. Several genes that have been implicated in myoblast fusion were aberrantly regulated during differentiation when FAK was inhibited. Intriguingly, the normal increases in the transcript of caveolin 3 as well as an integrin subunit, the beta1D isoform, were suppressed by FAK inhibition. We confirmed this also at the protein level and show that direct inhibition of beta1D subunit expression by siRNA inhibited myotube formation with a prominent effect on secondary fusion. These data suggest that FAK regulation of profusion genes, including caveolin 3 and the beta1D integrin subunit, is essential for morphological muscle differentiation.

TGF-β1 modulates focal adhesion kinase expression in rat intestinal epithelial IEC-6 cells via stimulatory and inhibitory Smad binding elements

TGF-beta and FAK modulate cell migration, differentiation, proliferation and apoptosis, and TGF-beta promotes FAK transcription in intestinal epithelial cells via Smad-dependent and independent pathways. We utilized a 1320 bp FAK promoter-luciferase construct to characterize basal and TGF-beta-mediated FAK gene transcription in IEC-6 cells. Inhibiting JNK or Akt negated TGF-beta-stimulated promoter activity; ERK inhibition did not block the TGF-beta effect but increased basal activity. Co-transfection with Co-Smad4 enhanced the TGF-beta response while the inhibitory Smad7 abolished it. Serial deletions sequentially removing the four Smad binding elements (SBE) in the 5' untranslated region of the promoter revealed that the two most distal SBE's are positive regulators while SBE3 exerts a negative influence. Mutational deletion of two upstream p53 sites enhanced basal but did not affect TGF-beta-stimulated increases in promoter activity. TGF-beta increased DNA binding of Smad4, phospho-Smad2/3 and Runx1/AML1a to the most distal 435 bp containing 3 SBE and 2 AML1a sites by ChIP assay. However, although point mutation of SBE1 ablated the TGF-beta-mediated rise in SV40-promoter activity, mutation of AML1a sites did not. TGF-beta regulation of FAK transcription reflects a complex interplay between positive and negative non-Smad signals and SBE's, the last independent of p53 or AML1a.

Fak56 functions downstream of integrin alphaPS3betanu and suppresses MAPK activation in neuromuscular junction growth

BackgroundFocal adhesion kinase (FAK) functions in cell migration and signaling through activation of the mitogen-activated protein kinase (MAPK) signaling cascade. Neuronal function of FAK has been suggested to control axonal branching; however, the underlying mechanism in this process is not clear.ResultsWe have generated mutants for the Drosophila FAK gene, Fak56. Null Fak56 mutants display overgrowth of larval neuromuscular junctions (NMJs). Localization of phospho-FAK and rescue experiments suggest that Fak56 is required in presynapses to restrict NMJ growth. Genetic analyses imply that FAK mediates the signaling pathway of the integrin αPS3βν heterodimer and functions redundantly with Src. At NMJs, Fak56 downregulates ERK activity, as shown by diphospho-ERK accumulation in Fak56 mutants, and suppression of Fak56 mutant NMJ phenotypes by reducing ERK activity.ConclusionWe conclude that Fak56 is required to restrict NMJ growth during NMJ development. Fak56 mediates an extracellular signal through the integrin receptor. Unlike its conventional role in activating MAPK/ERK, Fak56 suppresses ERK activation in this process. These results suggest that Fak56 mediates a specific neuronal signaling pathway distinct from that in other cellular processes.

Focal adhesion kinase: a promising target for anticancer therapy

Focal adhesion kinase (FAK) is a protein tyrosine kinase acting as an early modulator of the integrin signalling cascade, thus regulating various basic cellular functions. In transformed cells, upregulation of FAK protein expression and uncontroled signalling were held responsible for the promotion of malignant phenotypic characteristics, as well as resistance to chemotherapy and radiotherapy. Direct FAK targeting resulted in the inhibition of the malignant phenotype of cancer cells, whereas increased apoptotic rates of cancer cells, either used alone or in combination with conventional chemotherapeutic agents, radiotherapy or hormonal therapy. Furthermore, drugs used in cancer chemotherapy, besides their basic mode of action, were also shown to act through altering FAK signalling. Finally, positive results were noted by the transfection of cancer cells with fak mutants or genes that suppress FAK expression or activity, such as phosphatase and tensin homolog deleted on chromosome Ten (PTEN), ribonucleotide reductase M1 polypeptide (RRM1) and melanoma differentiation-associated gene-7 (mda-7). The purpose of this article is a comprehensive review of the existing data on the possible use of FAK targeting in anticancer therapy.

Overexpression of Focal Adhesion Kinase in Head and Neck Squamous Cell Carcinoma Is Independent of fak Gene Copy Number

The development of human malignancies can involve the aberrant regulation of intracellular signal transduction pathways that regulate cell-extracellular matrix interactions. In the current study, we aimed to evaluate focal adhesion kinase (FAK) at both genetic and protein expression levels in head and neck squamous cell carcinomas (HNSCC) and to explore the prognostic significance of FAK. A total of 211 tissue specimens, including 147 primary tumors, 56 lymph node metastases, 3 benign hyperplasias, and 5 dysplasias, were analyzed using immunohistochemistry. The fak gene dosage was determined in 33 tumors. Correlations among DNA, protein, and clinicopathologic variables were analyzed. FAK protein was overexpressed in HNSCCs compared with corresponding normal mucosa. High expression levels were found in 62% of the samples. Positive immunostaining was also detected in benign hyperplasias and preinvasive dysplastic lesions. All lymph node metastases examined showed FAK overexpression, with significant correlation with the expression in matched primary tumor. DNA copy number ratios for fak were higher in 39% of the tumors compared with normal mucosa. However, elevated FAK expression did not correlate with gains on DNA level, and not all cases with an amplification of the fak gene displayed protein overexpression. Similar data were obtained in five HNSCC-derived cell lines, in which FAK mRNA levels were precisely correlated with FAK protein levels. FAK protein overexpression in tumors correlated with nodal metastases. These findings suggest an involvement of FAK in the onset and progression of HNSCC and provide an insight into a mechanism of FAK activation alternative to gene amplification.

Quantitative kinetic analysis of gene expression during human osteoblastic adhesion on orthopaedic materials

Little information was found in the literature about the expression on hydroxyapatite (HA) materials of genes specific of cellular adhesion molecules although more were found on titanium-based substrates. Hence, the goal of this work was to study by a kinetic approach from 30 min to 4 days the adhesion of Saos-2 cells on microporous (mHA) and non-microporous hydroxyapatite (pHA) in comparison to polished titanium. Our strategy associated the visualization of adhesion proteins inside the cells by immunohistochemistry and the quantitative expression of genes at mRNA level by real-time PCR. The cell morphology was assessed using scanning electron microscopy and the number of cells thanks to biochemical techniques. The cellular attachment was the highest on mHA from 30 min to 24 h although the cell growth on mHA was the lowest after 4 days. Generally, the Saos-2 osteoblastic cells morphology on mHA was radically different than on other surfaces with the particularity of the cytoplasmic edge, which appeared un-distinguishable from the surface. The revelation by specific antibodies of proteins of the cytoskeleton (actin) and the focal adhesions (FAK, phosphotyrosine) confirmed that adhesion and spreading were different on the 3 materials. The actin stress fibres were less numerous and shorter on mHA ceramics. Cells had more focal contacts after 4 h on mHA compared to other substrates but less after 24 h. The highest values of total proteins were extracted from mHA at 0.5 and 24 h and from pHA at 1, 4, and 96 h. The αv and β1 integrin, actin, FAK, and ERK gene expression were found to be different with adhesion time and with materials. C-jun expression was comparable on mHA, titanium and plastic but was largely higher than on pHA at 0.5 and 1 h. On the contrary, c-fos expression was the highest on pHA after 0.5 h and the lowest after 1 h. This difference between c-fos and c-jun expression on pHA after 0.5 h could be related to the fact that these two genes may differ in their signalling pathways. The expression of the alkaline phosphatase gene after 4 days was lower on mHA compared to other materials demonstrating that the microstructure of the mHA ceramic was not favourable to Saos-2 cells differentiation. Finally, it was demonstrated in this study that HA and titanium surfaces influence as well gene expression at early times of adhesion as the synthesis of adhesion proteins but also proliferation and differentiation phases. Indeed, the signal transduction pathways involved in adhesion of Saos-2 cells on HA and titanium were confirmed by the sequential expression of αv and β1 integrins, FAK, and ERK genes followed by the expression of c-jun and c-fos genes for proliferation and alkaline phosphatase gene for differentiation.

Evaluation of Focal Adhesion Kinase Gene Expression in Leukemia and Cancer Cell Lines Using Combined Molecular Cytogenetic Investigations and Quantitative Real Time RT-PCR.

FAK (Focal Adhesion Kinase) is a non-receptor tyrosine kinase protein implicated in integrin-mediated signal pathways. The human gene fak is located on chromosome 8q near c-myc. Cells that overexpress FAK show increased migration and increased cell survival with apoptosis inhibition. The aim of the present study was to investigate the genomic or post-genomic origin of this overexpression in seven tumor cell lines with different levels of FAK expression: K562, an erythroblastic cell line; U937, a monoblastic cell line; KG1a, a poorly differenciated myeloblastic cell line; HL 60, a promyelocytic cell line; Jurkat, a lymphoblastic cell line; A549, a bronchial adenocarcinoma cell line; M4Beu, a lymph node metastasis melanoma cell line. Total or partial chromosome 8 qualitative or quantitative abnormalities and the presence of double minute chromosomes bearing coding genomic DNA fragments were investigated using both conventional cytogenetic (standard karyotype after R- and G- banding) and molecular cytogenetic such as multicolor karyotype (M-FISH), FISH and comparative genomic hybridization (CGH). The mRNA expression was investigated using quantitative real time RT-PCR and the protein level using immunocytochemistry and western blot (immunoblot) analysis. Except for the Jurkat cell line, karyotypes revealed complex modifications. FISH and M-FISH allowed to precisely identify the chromosomal origin of the genomic material on each chromosome and CGH allowed to characterize relative loss and gain of coding genomic DNA and to identify their chromosomal origin. Summarized results are shown in the following table:No detectable RT-PCR product was observed for HL60 and U937 cell lines. The preliminary results show that there was usually a good correlation between PCR data and western blot analysis. However, FAK overexpression for KG1a cell line most probably has a genomic origin, while in K562 and A549 cell lines the origin is most probably at the transcription or post-transcription level without any abnormality of chromosome 8. Dysregulation of fak gene is observed in hematopoietic as well as solid tulor cell lines, and may contribute to leukemogenesis.</CENTER>Cell lineFISH 8q24-qterCGH Chr. 8RT-PCRRT-Q-PCRWestern blotK562NormalNormal+130.1++++KG1aAmplifiedenh(8)(q11qter)+37.5++++JurkatNormalNormal+8.7+ (low)U937Normalenh(8)(pterqter)000HL60Amplifiedamp(8)(q23-24)000M4BeuNormalNormal+43.50A549NormalNormal+46++++RT-Q-PCR results in copy number ratio

Alterations in granule matrix and cell surface of focal adhesion kinase-deficient mast cells.

Focal adhesion kinase (FAK) is a nonreceptor protein tyrosine kinase that plays an important role in many cellular processes and is tyrosine phosphorylated after FcepsilonRI aggregation in mast cells. In mice, null mutation of the fak gene results in a lethal phenotype in which the embryos fail to develop past day 8.5 of gestation. To study the role of FAK in these mast cells, 8.5-day embryos were isolated and placed in culture with IL-3 and stem cell factor (SCF). Although FAK was not required for the development of mast cells in culture, the FAK(-/-) embryo-derived mast cells had several distinct characteristics. Compared with the controls, the mast cells that lack FAK were less metachromatic and by electron microscopy had granules that appeared largely electron lucid, although their histamine content was unchanged. The FAK-deficient mast cells had a reduction in the content of chondroitin/dermatan sulfate, the major glycosaminoglycan component of the granular matrix. The FAK-deficient cells had fewer microvilli that were fused with each other, giving the cell surface a ruffled appearance. There was also a 3-fold increase in the number of cells highly expressing beta(7) integrin. However, signal transduction from the high affinity IgE receptor for the secretion of histamine was similar in the wild-type, heterozygote, and the FAK-deficient cells. The FcepsilonRI-induced tyrosine phosphorylation of paxillin, Crk-associated tyrosine kinase substrate (CAS), and mitogen-activated protein kinase proteins was independent of FAK. These results indicate that FAK plays a role in regulating the glycosaminoglycan content of the secretory granules and influences the cell surface morphology of mast cells.

Rat oligodendroglia express c-met and focal adhesion kinase, protein tyrosine kinases implicated in regulating epithelial cell motility

Oligodendrocytes, the myelinating cells of the central nervous system, arise from a profilerating pool of motile progenitor cells. The proliferation and survival of these cells is dependent on signal transduction via several protein tyrosine kinases (PTKs) including receptors for fibroblast growth factor -2, the platelet-derived growth factor receptors and the neurotrophin receptor, trkC. We hypothesized that additional PTKs could also influence oligodendroglial development. Utilizing RTPCR, we amplified from post-natal day 6 rat oligodendroglia 17 distinct kinase domain sequences, 14 of which were not previously known to be expressed by oligodendroglia. Amongst the sequences identified were the c-met and Fak genes, whose protein products regulate the motility of other epithelial cell types. Utilizing immunohistochemistry, we confirmed that both c-met and Fak are expressed by cultured oligodendroglia, suggesting that these proteins could also be implicated in regulating the motility of these cells.

Increased dosage and amplification of the focal adhesion kinase gene in human cancer cells.

Focal adhesion kinase (pp125FAK) is present at sites of cell/extracellular matrix adhesion and has been implicated in the control of cell behaviour. In particular, as a key component of integrin-stimulated signal transduction pathways, pp125FAK is involved in cellular processes such as spreading, motility, growth and survival. In addition, a number of reports have indicated that pp125FAK may be up-regulated in human tumour cells of diverse origin, and consequently, a role has been proposed for pp125FAK in the development of invasive cancers. However, to date the mechanisms that lead to elevated pp125FAK expression in tumour cells have not been determined. Here we used in situ hybridization to confirm chromosome 8q as the genomic location of the human fak gene and report that elevation of pp125FAK protein in cell lines derived from invasive squamous cell carcinomas is accompanied by gains in copy number of the fak gene in all cases examined. In addition, we observed increased fak copy number in frozen sections of squamous cell carcinomas. Furthermore, increased dosage of the fak gene was also observed in many cell lines derived from human tumours of lung, breast and colon, including two cell lines Calu3 and HT29, in which fak was amplified. In addition, in an in vitro model for human colon cancer progression there was a copy number gain of the fak gene during conversion from adenoma to carcinoma, which was associated with increased pp125FAK protein expression. Thus, we show for the first time that many cell lines derived from invasive epithelial tumours have increased dosage of the fak gene, which may contribute to the elevated protein expression commonly observed. Although other genes near the fak locus are co-amplified or increased in copy number, including the proto-oncogene c-myc, the biological properties of pp125FAK in controlling the growth, survival and invasiveness of tumour cells, suggest that it may contribute to the selection pressure for maintaining increased dosage of the region of chromosome 8q that encodes these genes.

Suppression of Protein Kinase C Is Associated with Inhibition of PYK2 Tyrosine Phosphorylation and Enhancement of PYK2 Interaction with Src in Thrombin-Activated Platelets

Blood platelets have recently been shown to express PYK2, a nonreceptor tyrosine kinase belonging to the FAK gene family. In this study, we examined the involvement of protein kinase C (PKC) in PYK2-related responses in human platelets. While PYK2 tyrosine phosphorylation induced by thrombin was inhibited by preincubation of platelets with PKC inhibitors, staurosporine and Ro31-8220, PYK2 association with Src was markedly enhanced under the same conditions. Platelet intracellular Ca 2+ mobilization induced by thrombin was hardly inhibited by these PKC inhibitors. p130 Cas is a docking protein that associates with FAK or PYK2 through the SH3 domain. Although we identified p130 Cas in platelets for the first time, this docking protein failed to interact with PYK2. These results suggest that PKC activation (but not Ca 2+ mobilization) is involved in PYK2 tyrosine phosphorylation and that PYK2 associates with Src without PYK2 tyrosine phosphorylation or p130 Cas involvement in platelets.