Masters Theses

Date of Award

8-1990

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Carl J. Wust

Committee Members

A. Ichiki, R. Moore

Abstract

The detrimental effect of specific antibody on K-562 cells is evident in certain cellular processes. K-562 human leukemia cells grown in the presence of rabbit-anti-K-562 serum or derived gamma globulin die within 5 days without observable lysis. Within the first 24 hours, there is over a 90% decrease in DNA synthesis. These events occur in the absence of complement or effector cells. To characterize the mechanism of this induced cell death using well-defined antigens, 9 monoclonal antibodies found to be reactive with K-582 cells by cytofluorography, could be categorized into three groups based on their effect on the uptake of 3H-TdR into acid precipitable DNA. The groups are; no effect [IL-2R1(lgG2a), Leu-M1 (lgG2b), Leu-M1 (IgM), OK-M1 (lgG2b)], increased 3H-TdR uptake [BA-1(lgM), HLA-A,B,C(lgG2a)] and decreased 3H-TdR uptake [OKT9(IgG1), OKB2(lgG1), and PM81 (IgM)]. These categories are not correlated with antibody isotype, suggesting that a non-specific Fc binding event is not responsible for the antibody inducement of cell death. These findings suggested that antibody may induce a programmed cell death, or apoptosis. Since one of the main event markers for apoptosis is DNA fragmentation, this was assesed in the K-562 model. DNA fragmentation studied in most apoptotic systems has been done in the mouse and evidence of DNA degradation in human cells has been controversial. As expected, we did not find double strand fragmentation of DNA from K-562 cells treated with specific antiserum. Upon examination of DNA from K-562 cells treated with antiserum, extensive single strand nicking was observed after analysis of strand separating agarose electrophoresis and centrifugation of purified DNA through an alkaline (pH 13) sucrose gradient. Interestingly, the DNA of control K-562 cells also showed evidence of single strand nicking albeit to a lesser extent than treated cells. Antibody induced cell death could not be prevented with cycloheximide, suggesting that protein synthesis is not required for DNA cleavage. Antibody induced cell death was prevented by the addition of aurintricarboxylic acid, an endonuclease inhibitor and general inhibitor of DNA binding proteins. All of these results taken together support the conclusion that some surface receptors react with antibody to transduce signals which lead to a programmed cell death. Once the mechanisms of these transductions are understood, other reagents could be used to trigger the process. This capability to trigger death in unwanted cells could have a unique therapeutic advantage.

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