Date of Award

12-2001

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Albert T. Ichiki

Committee Members

Karla Matteson, Robert N. Moore, Carmen Lozzio

Abstract

The K-562 cell line was developed from the pleural effusion of a patient with chronic myelogenous leukemia (CML) in blast crisis and has served as an excellent model to study the properties of CML cells. The highly undifferentiated cells can be induced by a variety of chemicals to follow multiple pathways of differentiation. When K-562 cells were treated with phorbol myristate acetate (PMA), they exhibited accessory cell functions by replacing monocytes in the activation of resting Th2 lymphocytes. However, K-562 cells are negative for the expression of class II major histocompatibility complex (MHC) antigens HLA-DR, -DP, and -DQ. The focus of this study was to determine the functional capacity of the class II transactivator (CIITA), a crucial regulatory factor of MHC class II genes, in K-562 cells. CIITA is a co-activator, which is regulated in a tissue-specific manner by four alternative promoters. The transcription factor is recruited to the MHC class II promoter by factors that associate with the promoter, such as regulatory factor X (RFX). The CIITA mRNA is present in K-562 cells and, due to alternative splicing, there is an insertion of an additional genomic sequence not present in the wild-type. The insertion occurs at the carboxy terminus of the CIITA gene, introducing a stop codon at nucleotide 2796, which results in a truncated protein of 932 rather than 1130 amino acids. Although a truncation of this type does not interfere with the ability of CIITA to associate with the MHC class II promoter or with the RFX complex in vivo, it does lead to an inactive protein, which might explain the absence of MHC class II molecules on K-562 cells. The most active CIITA promoter in these cells is promoter III, which is generally specific to B cells. Although IFN-g treatment of K-562 cells does not result in MHC class II expression, the IFN- g inducible CIITA promoter IV does exhibit low levels of basal activity, and the activity of promoter IV is greatly enhanced upon treatment with IFN- g. These results provide insight into the role of CIITA in malignant cells, as well as supporting the hypothesis that the negative expression of MHC class II molecules is caused by the alternatively spliced CIITA transcript identified in K-562 cells.

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