Clonogenicity of Human Promyelocytic Leukemia Cells Enlarged and Multinucleated by Treatment with Cytochalasin B and their Potential as Targets for Improved Chemical and Physical Therapeutic Approaches
Honors Capstone Project
Date of Submission
Dr. Thomas Fondy
Dr. John Belote
Arts and Science
Capstone Prize Winner
Won Capstone Funding
Sciences and Engineering
Cytochalasin B (CB) is a pharmacological agent produced by fungi that disrupts the actin microfilament cytoskeleton and inhibits cytokinesis. It interferes with the formation of the contractile ring and with the development of the cleavage furrow, thus preventing cell division. When normal cells are treated with CB, cell cycle arrest occurs. However, when neoplastic cells are treated with CB, the cells continue to enter the cell cycle and to make new nuclei, but since cytokinesis cannot occur, the neoplastic cells become enlarged and multinucleated. Our lab proposes that enlarged multinucleated neoplastic cells might be more susceptible to agents damaging DNA because of the excess number of nuclear targets per cell. Since normal cells exit the cell cycle when treated with CB, they should not show increased sensitivity to DNA damaging agents but rather should be protected from such damage because they would be resting cells.
Wells in 96-well optically clear bottomed plates are seeded with 5 ul of cells and sterile polystyrene beads. The number and size of cells and the number of beads per well are determined visually with a Reichert compound microscope. After recording the well contents, the wells are filled with 20% FBS growth medium and the plates are covered with a sterile gas permeable membrane. The plates are allowed to clone in a carefully humidified incubator for two weeks. Wells containing cloned leukemia cells are observed after two weeks and the wells with clonogenic cells are correlated with the contents of those wells originally recorded.
In an alternative procedure, cells and beads are mixed in agarose medium and seeded into culture flasks. The congealed agarose retains the cells and beads in their initial positions. The flasks are inverted for viewing with the Reichert microscope. The positions and sizes of cells in the agarose medium are recorded on a polar graph paper. The flasks are covered with additional agarose medium and allowed to clone for two weeks. The positions of clones in the agarose layer are correlated with the size of cells recorded at that position at the time of flask seeding.
I seeded sixteen 96 well plates in that could be evaluated in eleven separate experiments and I also seeded three culture flasks. The sixteen plates contained 2423 cells in 1199 wells (wells with contents that hit the edges or mold contamination are eliminated from counting). Of the 2423 cells 94% were 19 u or greater in size, and 5% to 10% of these were observed to be clonogenic. The 6% of cells that remained smaller than 19 u were observed to have very low cloning efficiency (4%) indicating that they were non-clonogenic dead cells.
These procedures allow me to establish that a significant proportion of the enlarged CB-treated U937 cells remain clonogenic. Cloning the CB-treated cells in soft agarose allows me to determine whether CB-treatment induces differentiation in the U937 cells because we can directly observe the type of colony being formed by each clonogenic cell. This work shows the enlarged multinucleated CB-treated U937 cells retain viability and clonogenicity when the CB is diluted out and thus remain potentially pathogenic and are targets for therapeutic modalities based on increased size and multinucleation.
Dubois, Shannon, "Clonogenicity of Human Promyelocytic Leukemia Cells Enlarged and Multinucleated by Treatment with Cytochalasin B and their Potential as Targets for Improved Chemical and Physical Therapeutic Approaches" (2007). Syracuse University Honors Program Capstone Projects. 565.
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