Role of MSH6 mutations in North American patients receiving clinical genetic testing for hereditary nonpolyposis colorectal cancer
Creators: name: Andersen, Melissa
Publisher: name: University of Utah
Year: 2012
Subjects: subject: Colon Cancer
subject: Genetics
subject: FOS: Biological sciences

description: The relationship of fibrinolytic activity produced by baby hamster kidney (BHK-21) cells to surface properties and growth of the cells has been studied.  Hydrolysis of fibrin clots and casein was used to detect cellular protease activity.  Cloned, high passage sublines of the BHK-21/C13 cell line were found to produce increased fibrinolysis when compared to low passage BHK-21/C13 cells (C13).  Low passage C13 cells were also found to produce an inhibitor of the cellular fibrinolysin.  This inhibitory activity was detected when low and high protease-producing cell lines were co-cultivated and could be removed from the C13 cells by trypsin treatment.  Inhibitor could again be detected in C13 cell cultures after 6-8.5 hours incubation at 37°C following typsinization.  This substance was not detected in supernatant fluids from growing C13 cells and its production was inhibited by incubation of cells at 4°C.  All high protease-producing cultures formed colonies in agar suspension, exhibited quantitative increases in agglutinability by  concanavalin A (Con A) when compared to low passage cell cultures and grew faster as judged by transfer behavior of cells and increased rates of accumulation of cells in metaphase when grown in the presence of mitotic inhibitor.  Low protease-producing C13 cells and sublines low in protease activity isolated from high passage cultures did not grow in agar suspension, exhibited slower growth rates and decreased agglutinability by Con A.  Low protease-producing C13 cells could be stimulated to produce increase amounts of fibrinolytic activity with accompanying changes in surface properties characteristic of transformed cells by addition of purines such as hypoxanthine or adenosine to culture media.  The effect was reversed by deletion of the purines.  Growth of C13 cell in agar suspension did not occur with purine supplementation alone.  With the addition of hypoxanthine and thymidine or adenosine and thymidine or adenosine and thymidine, C13 cells formed colonies in agar suspension which were similar to those formed by high protease-producing cell lines.  Fibrinolytic activity could be detected from the colonies formed by C13 cells in agar suspension.  Treatment of C13 cells suspended in agar with trypsin or fibrinolytically active culture fluids from high protease-producing cells did not result in the multiplication of the cells in agar.  Temperature sensitive BHK-21 cells were isolated which were defective for cytokinesis but not karyokinesis.  At 39-40°C these cells became enlarged and multinucleated.  The defect did not appear to be associated with the cellular fibrinolysin because protease activity could be detected at the permissive and non-permissive temperatures.  Growth of temperature sensitive cells in agar suspension did not occur at 39-40°C but cell enlargement, vacillation and lysis were observed in a large portion of the cell population.  The data demonstrate that growth in agar suspension, increased Con A agglutinability and increased growth rates are positively associated in some way with the detection of increased fibrinolysis.  An increase in the amount of fibrinolysin.produced by high passage BHK-21 cultures was always associated with increase Con A agglutinability of cells.  Although increased fibrinolytic activity could always be detected from cells which grew in agar suspension, and increase in protease synthesis was not always followed by growth in agar suspension.  This suggests that factors in addition to the fibrinolysin produced by cell are involved in growth regulation.