apyrase and 1-10-phenanthroline

Tumour cells activate and aggregate platelets [tumour cell-induced platelet aggregation (TCIPA)] and this process plays an important role in the successful metastasis of cancer cells. To date, most studies on TCIPA have been conducted under no-flow conditions. In this study, we have investigated TCIPA in real time under flow conditions, using an ultrasound standing wave trap that allows formation and levitation of cancer cell clusters in suspension, thus mimicking the conditions generated by flowing blood.. Using 59M adenocarcinoma and HT1080 fibrosarcoma cells and human platelets, cancer cell cluster-platelet aggregates were imaged in real time using epi-fluorescence microscopy (F-actin) and investigated in detail using confocal microscopy (matrix metalloproteinase-2-GPIIb/IIIa co-localization) and scanning electron and helium-ion microscopy (<1 nm resolution). The release of gelatinases from aggregates was studied using zymography.. We found that platelet activation and aggregation takes place on the surface of cancer cells (TCIPA), leading to time-dependent disruption of cancer cell clusters. Pharmacological modulation of TCIPA revealed that EDTA, prostacyclin, o-phenanthroline and apyrase significantly down-regulated TCIPA and, in turn, delayed cell cluster disruption, However, EGTA and aspirin were ineffective. Pharmacological inhibition of TCIPA correlated with the down-regulation of platelet activation as shown by flow-cytometry assay of platelet P-selectin.. Our results show for the first time, that during TCIPA, platelet activation disrupts cancer cell clusters and this can contribute to metastasis. Thus, selective targeting of platelet aggregate-cancer cell clusters may be an important strategy to control metastasis.

Topics: Actins; Adenocarcinoma; Apyrase; Blood Platelets; Cell Communication; Cell Line, Tumor; Down-Regulation; Edetic Acid; Epoprostenol; Female; Fibrosarcoma; Flow Cytometry; Humans; Matrix Metalloproteinase 2; Microscopy, Confocal; Microscopy, Fluorescence; Ovarian Neoplasms; P-Selectin; Phenanthrolines; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Tumor Cells, Cultured; Ultrasonics; Ultrasonography

The two transmembrane domains flanking the active site of CD39 regulate its activity, but little is known about the structural and dynamic features underlying their importance. Here we use a disulfide crosslinking strategy to examine transmembrane helix interactions and dynamics and to correlate these features with activity and substrate binding. We find strong intrasubunit TM1-TM2 interactions, as well as TM1-TM1' and TM2-TM2' interactions between dimer subunits, near the extracellular side of the membrane but only weak interactions near the cytoplasmic end. The specific helix faces that constitute each interface are highly flexible, indicating a significant degree of rotational mobility within the packed structure. Analysis of activity after locking the helices in various orientations via disulfide bonds suggests that not only the arrangement but also the ability of the helices to move relative to each other is crucial for enzyme function. Helix mobility is in turn modulated by substrate binding. These results suggest that rather than playing a static structural role to support an optimal active site conformation, the transmembrane domains undergo dynamic motions that underlie their functional relationship with the active site.

Topics: Adenosine Triphosphatases; Amino Acid Substitution; Animals; Antigens, CD; Apyrase; Binding Sites; COS Cells; Cross-Linking Reagents; Cysteine; Dimerization; Enzyme Activation; Enzyme Stability; Mutagenesis, Site-Directed; Phenanthrolines; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Rats; Substrate Specificity; Thermodynamics; Transfection