Marianna Gyenes

Distribution of Gs-α activating mutations in human thyroid tumors measured by subcloning

In the search for the prevalence and distribution pattern of Gs-α gene mutations in differentiated thyroid tumors we examined 66 tumor tissue samples for the presence of mutations at “hot-spot” codons 201 and 227 using methods based on the polymerase chain reaction, subcloning and sequencing. the prevailing type of singlebase substitution at codon 201 (71.4%) corresponded to the replacement of the wild-type sequence CGT (Arg) with TGT (Cys). The fragments of the Gs-α gene, including codon 201 or 227 from five follicular carcinomas and one follicular adenoma, were subcloned inEscherichia coli and it was found that the proportion of alleles with mutated codon 201 varied from 3.2% to 43%. Sequencing of the corresponding region has confirmed preliminary data indicating that the single-base changes CGT (Arg) to TGT (Cys) or CGT to CAT (His) occurred. There was only a weak correlation between the prevalence of cells bearing a mutation in the Gs-α gene and the level of Gs-α protein expression in the corresponding thyroid tumors.

Fibronectin unfolded by adherent but not suspended platelets: An in vitro explanation for its dual role in haemostasis

Fibronectin (FN), a dimeric adhesive glycoprotein, which is present both in plasma and the extracellular matrix can interact with platelets and thus contribute to platelet adhesion and aggregation. It has been shown that FN can decrease platelet aggregation but enhance platelet adhesion, suggesting a dual role of FN in haemostasis. The prevalent function(s) of FN may be determined by its fibril form. To explore the suggested dual role of this adhesive protein for haemostasis in further detail, we now tested for any differences of adherent and suspended platelets with regard to their effect to unfold and assemble FN upon interaction. Platelet aggregation and adhesion assays were performed using washed platelets in the presence of exogenous FN. Addition of plasma FN reduced platelet aggregation in response to collagen or PMA by 50% or 25% but enhanced platelet adhesion onto immobilized collagen, as compared to control experiments. Analyses by fluorescence resonance energy transfer (FRET) demonstrated that adherent platelets but not suspended platelets were capable of unfolding FN during 3h incubation. Fluorescence microscopy and deoxycholate (DOC) solubility assays demonstrated that FN fibrils formed only on the surfaces of adherent platelets. In addition, platelets adherent onto FN revealed a significantly higher activity of specific Src phosphorylation (pY418) than platelets in suspension. These data suggest (1) that the function of FN in haemostasis is prevalent to its assembly, unfolding and subsequent fibril formation on the surface of adherent platelets and (2) that outside-in signaling contributes to the interaction of platelets and FN.

Impact of shear stress on Src and focal adhesion kinase phosphorylation in fibrinogen-adherent platelets.

&NA; Shear stress alone can activate platelets resulting in a subsequent platelet aggregation, so-called ‘shear-induced platelet aggregation’. In our work, we analyzed how differently elevated shear stress impacts the Src and focal adhesion kinase (FAK) activation in fibrinogen-adherent human platelets. We detected the extents of Src pY418 and FAK pY397 activations in platelets on immobilized fibrinogen and over BSA under shear conditions. Moreover, we analyzed the role of &agr;IIb&bgr;3 in the shear-induced platelet signaling by performing our experiments in the presence of the &agr;IIb&bgr;3-antagonist Abciximab. Abnormally high shear rates (5000 s−1) significantly increased the extent of phosphorylation of both tyrosine kinases after short (2 min) incubation time independently of the presence or absence of the integrin &agr;IIb&bgr;3 ligand, fibrinogen. We could see considerably greater Src activation on immobilized fibrinogen than on BSA, but the extent of FAK Y397 phosphorylation was independent on the matrix. Abciximab not only reduced the Src and FAK signaling in platelets exposed to 5000 s−1 on immobilized fibrinogen, but in platelets exposed to 5000 s−1 over BSA as well. Our data indicate that whereas Src activation under shear stress is dominantly ligand-dependent, FAK signaling seems to be mostly shear induced.

Integrin αIIbβ3-Dependent ERK Signaling Is Regulated by Src and Rho Kinases in Both Leu33 and Pro33 Polymorphic Isoforms

Platelet integrin αIIbβ3 possesses a Leu/Pro polymorphism at residue 33 (Leu33/HPA-1a or Pro33/HPA-1b). The Pro33 isoform has been suggested to exhibit prothrombotic features. αIIbβ3-expressing CHO (Chinese hamster ovary) cells on immobilized fibrinogen show activation of the MAP kinase family member ERK2, with an enhanced ERK2 activity in Pro33 cells compared to Leu33 cells. In our present work, we examined how the Leu/Pro polymorphism modulates the ERK2 activation stimulated by 2 differently triggered outside-in signalings. We either treated the CHO cells with Mn2+ or allowed them to adhere to fibrinogen. Moreover, we studied which signaling cascades are involved in ERK2 activation. In contrast to immobilized fibrinogen, Mn2+ did not significantly increase ERK2 activation. However, Mn2+ had a synergistic effect on ERK2 phosphorylation when combined with immobilized fibrinogen. Pro33 cells adherent to fibrinogen exhibited a significantly greater ERK2 activity than Leu33 cells in the presence of Mn2+, which peaked after 10 min of adhesion. Our data showed that Src family and rho kinases play a crucial role in the integrin αIIbβ3-dependent outside-in signaling to ERK2.

Molekularbiologische Charakterisierung von Tumoren durch frühzeitige Identifizierung von Mutationen in Onkogenen und Tumorsuppressorgenen

Die Transformation von normalen zu malignen Zellen schliest eine Kombination verschiedener genetischer Veranderungen im Sinne einer Mehrschritt-Karzinogenese ein, von denen heute schon einige mit entscheidender Bedeutung fur die Prognose der Patienten erkannt sind (z.B. ras, GSP, p53). Eine fruhzeitige Erkennung von potentiell malignen oder zunehmend entdifferenzierten Zellen in einem heterogenen Tumorgewebe (Adenom zu Karzinom) scheiterte bisher jedoch an der geringen Sensitivitat verwendeter molekular-biologischer Untersuchungsmethoden (mutationsspezifische Oli-gonukleotidhybridisierung (MOH), direkte Sequenzierung (DS)). Wir suchten deshalb nach Methoden, die bei ausreichend hoher Sensitivitat Mutationen aufdecken und damit Eingang in die klinische Diagnostik finden konnen.