Summary. Background: Tissue factor (TF)-bearing microparticles (MP) from different origins are thought to be involved in the pathogenesis of cancer-associated thrombosis. However, the role of circulating tumor cell-derived TF is not well understood. Methods: TF antigen and activity were measured in MP generated in vitro from human TF-expressing cancer cells by ELISA and clotting or thrombin generation assays, respectively. TF antigen and activity were also measured in vivo in cell-free plasmas from mice previously injected with in vitrogenerated MP or in cell-free plasmas from nude mice bearing orthotopically injected human cancer cells. Results: Tumor cellderived MP (TMP) exhibited strong TF-dependent procoagulant activity (PCA) in vitro and in vivo. Injection of TMP into mice was associated with acute thrombocytopenia and signs of shock, which were prevented by prior heparinization. Human TF antigen and activity could be detected in mouse cell-free plasmas up to 30 min after TMP injections. Human TF was detected in the spleen of injected mice and its clearance from circulation was delayed in splenectomized mice, suggesting the involvement of the spleen in the rapid clearance of circulating MP in vivo. Detectable levels of TF-dependent PCA and thrombin-antithrombin complex were found in cell-free plasmas from mice growing pancreatic human tumors, suggesting that circulating tumor-derived TF causes coagulation activation in vivo. Conclusions: MP derived from certain cancer cells exhibit TF-dependent PCA both in vitro and in vivo. These results provide new information about the specific contribution of tumor-derived MP to the hypercoagulable state observed in cancer.

Since little is known of how the primitive protozoan parasite, Giardia lamblia, senses and responds to its changing environment, we characterized a giardial protein kinase A (gPKA) catalytic subunit with unusual subcellular localization. Sequence analysis of the 1080-base pair open reading frame shows 48% amino acid identity with the cyclic AMP-dependent kinase from Euglena gracilis. Northern analysis indicated a 1.28-kilobase pair transcript at relatively constant concentrations during growth and encystation. gPKA is autophosphorylated, although amino acid residues corresponding to Thr-197 and Ser-338 of human protein kinase A (PKA) that are important for autophosphorylation are absent. Kinetic analysis of the recombinant PKA showed that ATP and magnesium are preferred over GTP and manganese. Kinase activity of the native PKA has also been detected in crude extracts using kemptide as a substrate. A myristoylated PKA inhibitor, amide 14-22, inhibited excystation with an IC 50 of 3 M, suggesting an important role of gPKA during differentiation from the dormant cyst form into the active trophozoite. gPKA localizes independently of cell density to the eight flagellar basal bodies between the two nuclei together with centrin, a basal body/centrosome-specific protein. However, localization of gPKA to marginal plates along the intracellular portions of the anterior and caudal pairs of flagella was evident only at low cell density and higher endogenous cAMP concentrations or after refeeding with fresh medium. These data suggest an important role of PKA in trophozoite motility during vegetative growth and the cellular activation of excystation.

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Prostate cancer is a complex and heterogeneous disease, and it is the most prevalent form of cancer and the second leading cause of cancer death in men (1). Along with the classic methods of diagnosing and prognosticating prostate cancer, utilizing stage, grade, and serum prostate-specific antigen level, molecular markers of aggressive disease are currently being evaluated as adjuncts to improve identification of tumors associated with a poor outcome. Because abnormal regulation of the cell cycle is one of the major causes of tumorigenesis in recent years, major advances have been made to understand the molecular mechanisms of aberrant cell cycle control leading to the development and progression of tumors. Studies on the aberrant expression of cell cycle regulatory genes in prostate carcinoma have identified a cdk 1 inhibitor p27 kip1 (2) and p53 (3) as biomarkers. These markers show a strong correlation with a high, moderate, or low risk of occurrence of prostate cancer. Immunohistochemical analysis of other G 1 /S check point-controlling proteins, including cyclins (4), the cdk inhibitor p16 (5), and retinoblastoma (6), have also been found to be helpful in identifying patients with aggressive prostate cancer.One of the major underlying mechanisms of aberrant cell cycle progression is uncontrolled DNA replication irrespective of cellular signals as a result of G 1 /S checkpoint failure. DNA replication is a highly ordered event controlled by sequential binding to and release of a number of replication proteins from the DNA sequence close to the origin of DNA replication. This also restricts firing of the replication origin to once per cell cycle. The regulation of initiation of DNA replication has been studied extensively in Saccharomyces cerevisiae, whereas understanding of this regulation in the mammalian system has just begun. However, identification of human homologs of yeast replication proteins, such as origin recognition complex (7-10), CDC6 (11), and minichromosome maintenance proteins (MCM) (12, 13), suggests evolutionarily conserved replication machinery. Studies on DNA replication in S. cerevisiae and Xenopus laevis have established a critical role of CDC6 in the initiation of replication (14 -16). In late mitosis, when mitotic cyclin (cyclin B) and cdk are inactive, CDC6 helps to establish the prereplicative complex (Pre-RC) on the chromatin-bound origin recognition complex, a six-subunit initiator protein (17), by recruiting MCM proteins and other factors (18 -20). In middle to late G 1 and at the G 1 /S transition, S-phase cyclins (cyclin E and cyclin A) and cdk2 are expressed (21,22) and initiate DNA replication by phosphorylating CDC6, which causes it to dissociate from Pre-RC (23). Release of CDC6 from chromatin and its translocation to the cytoplasm triggers initiation of DNA replication (24). Another role of CDC6 in S. cerevisiae is to function as an inhibitor of the G 2 /M phase until the S phase is complete (25). This defines CDC6 as a unique and critical protein with a dual role in regul...

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