Sensitivity of flow cytometric assay for measurement of human intracellular heat shock protein 27.
Increased expression of heat shock protein 27 (hsp27) is related to enhanced resistance of breast tumor cells to cytotoxic drugs and radiation therapy. Therefore, development of a rapid and sensitive method for detection of hsp27 may be useful for correlating tumor cell expression of hsp27 to breast cancer patients' clinical outcome. We have simultaneously assessed hsp27 levels in three different human cell lines (MCF-7, MDA-MB-231, and Jurkat) by both Western blotting and flow cytometry. MCF-7 hsp27 levels were consistently detected at higher levels, while MDA-MB-231 hsp27 levels were detected at very low levels when immunoblotting was performed. Hsp27 was not detected in Jurkat cells by immunoblotting. In contrast, hsp27 levels were detected by flow cytometry in all the cell lines, indicating a better sensitivity of this method. Although hsp27 was expressed in almost equal percentage of MCF-7 (93+/-3.4%), MDA-MB-231 (97+/-1%), and Jurkat (95.5+/-1.9) cells, the fluorescence intensity of intracellular hsp27 protein was significantly lower in MDA-MB-231 and Jurkat cells as compared to MCF-7 cells. The flow cytometry data further demonstrated that reduced hsp27 expression in both MDA-MB-231 and Jurkat cells was not due to a lack of hsp27 expression in a subset of cells, but rather due to reduced expression of hsp27 in all individual cells.
De
Asit K
AK
Department of Surgery, University of Rochester Medical Center Rochester, NY 14642, USA. asit_de@urmc.rochester.edu
Strickland
Jennifer
J
D015153:Blotting, Western; D045744:Cell Line, Tumor; D005434:Flow Cytometry / Q000379:methods*; D006360:Heat-Shock Proteins / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D012680:Sensitivity and Specificity
D006360:Heat-Shock Proteins