Colon cancer chemopreventive drugs modulate integrin-mediated signaling pathways.

Epidemiological studies of colorectal cancer incidence suggest that the development of this disease can be modulated by dietary factors. Among the micronutrients showing significant efficacy in tumor prevention are polyphenolic antioxidants found in fruits and vegetables. Epidemiological studies also indicate that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of colorectal cancer. Integrin-mediated cell-matrix contact provides critical signaling that regulates cellular proliferation, migration, and apoptosis. A signaling mediator for this system is focal adhesion kinase (FAK). Thus far, FAK has not been identified as a target for the inhibitory action of any chemopreventive drug in vivo or in vitro. However, the loss of integrin-mediated cell-matrix contact can induce apoptosis (anoikis), and effective chemopreventive agents typically increase the rate of enterocyte apoptosis. Therefore, we asked whether the NSAID, sulindac sulfide, and the phenolic antioxidant, caffeic acid phenethyl ester (CAPE), affected FAK expression or tyrosine phosphorylation in human colon carcinoma cells. We show that subapoptotic doses of both sulindac sulfide and CAPE caused a rearrangement of the actin cytoskeleton and consequently the loss of focal adhesion plaques. These drugs also reduced the tyrosine phosphorylation of FAK and an associated factor, p130Cas. Steady-state levels of these proteins, together with other relevant signaling molecules, remained unchanged after treatments. Finally, we show that both CAPE and sulindac reduced cell invasion, a functional assay for the inhibition of signaling downstream of FAK. These data strongly suggest that chemopreventive drugs can regulate FAK activity. In conclusion, these novel studies add modulation of integrin-mediated signaling to the spectrum of activity of NSAIDs and plant phenolics.

Selective association of the tyrosine kinases Src, Fyn, and Lyn with integrin-rich actin cytoskeletons of activated, nonaggregated platelets.

Integrin-mediated interactions between cytoskeletal proteins and extracellular fibrinogen are required for platelet adhesion. We have previously demonstrated that the major platelet integrin, alpha(IIb)beta(3), becomes incorporated into the actin cytoskeleton of platelets in an activation-dependent, aggregation-independent manner. To determine if regulatory molecules are also associated with these integrin-rich cytoskeletal complexes, we examined actin cytoskeletons for the presence of kinases and phosphoproteins. Western immunoblot analysis revealed that the tyrosine kinases Src, Fyn, and Lyn are specifically associated with actin cytoskeletons of activated, nonaggregated platelets. However, as noted by others, the cytoskeletal association of focal adhesion kinase depends on platelet aggregation. Actin cytoskeletons isolated from (32)P-labeled platelets also contain a number of phosphorylated proteins. Interestingly, an approximately 18-kDa phosphoprotein was uniquely present in activated platelet cytoskeletons. Collectively, our results demonstrate that actin cytoskeletons of activated, nonaggregated platelets contain not only integrins, but also kinases and phosphoproteins that could regulate platelet adhesion and transmembrane communication.

Endoscopic ultrasound for staging esophageal cancer, with or without dilation, is clinically important and safe.

BACKGROUND: To fully evaluate patients with esophageal cancer by endoscopic ultrasonography (EUS), the transducer must pass through the entire tumor to the cardia to scan the celiac axis. Dilation may be necessary. Published information suggests that dilation with EUS carries a sizeable risk. METHODS: In order to assess the complication rate associated with dilation prior to EUS in patients with esophageal cancer and the clinical significance of dilation for complete EUS staging, we reviewed the records of all patients who had undergone EUS for esophageal cancer. RESULTS: Sixty-three patients underwent EUS staging of esophageal cancer. Thirty-nine (62%) had lesions through which the EUS scope was passable (Group I). Ten (16%) patients (Group II) had lesions through which an EUS scope (diameter 13 mm) was unable to pass even after dilation. Fourteen patients (22%) had lesions that were dilated to allow passage of the EUS scope (Group III). All patients in Groups II and III had confirmation of EUS staging by CT and/or surgery. In Group II, five patients had tumors defined as T4 (50%) and five as T3 (50%). In Group III, nine (64%) had T4 tumors, four (29%) had T3, and one (7.7%) had T2. No complications were encountered in any group. CONCLUSION: EUS, either alone or after dilation, is a safe procedure and the complete EUS examination with celiac node visualization adds prognostically significant information.

Talin distribution and phosphorylation in thrombin-activated platelets.

We have previously demonstrated that the subcellular distribution of the adhesion plaque protein, talin, changes dramatically in human platelets in response to platelet activation (Beckerle et al., J. Cell Biol. 109, 3333-3346, 1989). Talin is uniformly distributed throughout the cytoplasm of resting platelets. However, when platelets are stimulated to become activated and adhesive, a significant amount of the talin population rapidly redistributes to a peripheral, submembranous location. In the present study we have examined talin phosphorylation and proteolytic cleavage as possible mechanisms by which talin's subcellular distribution could be regulated in platelets. We have found that thrombin activation of platelets leads to a fourfold increase in talin phosphorylation. Proteolytic cleavage of talin, however, is not detected in washed platelets activated with thrombin for as long as 30 minutes. Because talin moves to a submembranous location upon platelet activation and has been shown to interact with integrins in vitro, we also investigated whether the major platelet integrin, GPIIb-IIIa, is required for talin redistribution. Using Glanzmann thrombasthenic platelets, which are deficient in GPIIb-IIIa, we found that talin redistribution occurs even in the absence of GPIIb-IIIa. Collectively, our studies suggest that neither proteolytic cleavage of talin nor interactions between talin and GPIIb-IIIa is required for the regulated redistribution of talin in thrombin-activated platelets. Phosphorylation of talin in response to thrombin activation may, however, be one mechanism utilized by platelets to regulate talin distribution and function in human platelets.

Evidence for the selective association of a subpopulation of GPIIb-IIIa with the actin cytoskeletons of thrombin-activated platelets.

Activation of blood platelets triggers a series of responses leading to the formation and retraction of blood clots. Among these responses is the establishment of integrin-mediated transmembrane connections between extracellular matrix components and the actin cytoskeleton of the platelet. Here we report that a specific subpopulation of the major platelet integrin, glycoprotein IIb-IIIa (GPIIb-IIIa) (also referred to as alpha IIb beta 3 integrin), becomes incorporated into the detergent-insoluble actin cytoskeleton of platelets during the platelet activation response. The cytoskeletal association of GPIIb-IIIa is independent of platelet aggregation and fibrin sedimentation and is sensitive to cytochalasin D treatment. As determined by Western immunoblot analysis, approximately 22% of the total cellular GPIIb-IIIa becomes associated with the actin cytoskeleton upon thrombin activation in a manner that is independent of the detection of talin, alpha-actinin, or vinculin in the complex. We found that the cytoskeleton-associated GPIIb-IIIa is derived from an intracellular source since it is not available for lactoperoxidase-catalyzed radioiodination before platelet activation. Two intracellular sources of GPIIb-IIIa are present in resting platelets: GPIIb-IIIa associated with the alpha-granule secretory compartment as well as surface-inaccessible domains of the surface-connected canalicular system. Interestingly, alpha-granule secretion, which occurs in thrombin-activated platelets and results in the translocation of intracellular GPIIb-IIIa to the plasma membrane, appears to be required for the cytoskeleton incorporation of GPIIb-IIIa that we observe. Collectively, our data provide evidence that a subpopulation of GPIIb-IIIa derived from an intracellular source is selectively linked to the actin cytoskeleton of platelets upon thrombin activation in the absence of platelet aggregation.

Activation-dependent redistribution of the adhesion plaque protein, talin, in intact human platelets.

Talin is a high molecular weight protein localized at adhesion plaques in fibroblasts. It binds vinculin and integrin and appears to participate in generating a transmembrane connection between the extracellular matrix and the cytoskeleton. We have recently shown that talin is an abundant protein in platelets, cells highly specialized for regulated adhesion. Although talin constitutes greater than 3% of the total protein in intact human platelets, its location within the cells had not been defined. In the work reported here, we have investigated the distribution of talin in resting and activated human platelets by immunofluorescence and immunoelectron microscopy. We have found that talin undergoes an activation-dependent change in its subcellular location. In resting platelets, which are nonadhesive, talin is uniformly distributed throughout the cytoplasm. In contrast, in thrombin- and glass-activated, substratum-adherent platelets, talin is concentrated at the cytoplasmic face of the plasma membrane. This dramatic, regulated redistribution of talin raises the possibility that talin plays a role in the controlled development of platelet adhesion.

Use of endoscopic ultrasound in patients with esophageal motility disorders.

New technology has combined the endoscope with ultrasound in an effort to enhance the visualization of the gastrointestinal tract. With a modified standard endoscope that has an ultrasound transducer built into the tip, high frequency ultrasonic beams can be targeted in close proximity to existing lesions. This results in better quality resolution which enhances the evaluation of the targeted lesion. In addition, esophageal wall thickness can be evaluated and assessed as to its role in esophageal function.