Document Type

Honors Capstone Project

Date of Submission

Spring 5-1-2007

Capstone Advisor

Dr. Thomas Fondy

Honors Reader

Dr. John Belote

Capstone Major

Biology

Capstone College

Arts and Science

Audio/Visual Component

no

Capstone Prize Winner

no

Won Capstone Funding

no

Honors Categories

Sciences and Engineering

Subject Categories

Biology

Abstract

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.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

Included in

Biology Commons

Share

COinS
 
 

To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.