Isolation of Proteins Cross-linked to DNA by Cisplatin
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One way of identifying and further characterizing transcription factors is to study their association with DNA in situ. Many studies have performed this task using agents such as formaldehyde that crosslink proteins to DNA. However, the treatment of cells with agents such as formaldehyde results in the cross-linking of protein to DNA, and protein to protein. Thus, proteins cross-linked to DNA binding proteins may be misinterpreted as DNA binding proteins. To overcome this obstacle, researchers have focussed their attention on cisplatin (cis-DDP; cis-platinum (II)diamminedichloride), a cross-linking agent shown to crosslink protein to DNA and not to protein (1). Recent studies have shown that the majority of proteins cross-linked to DNA by cisplatin in situ are nuclear matrix proteins (2-4). We have also shown that cisplatin cross-links nuclear matrix-associated transcription factors and cofactors to DNA in the MCF-7 human breast cancer cell line (5). Thus, cisplatin appears to be an effective cross-linking agent for studying the role of transcription factors and nuclear matrix proteins in transcription. In support of this, we have discovered cisplatin DNA-cross-linked nuclear matrix (NM) proteins whose levels vary between well and poorly differentiated human breast cancer cell lines (6). Such changes in protein levels indicate that breast cancer development most likely involves changes in DNA organization, and, likely, changes in transcriptional events. Currently used nuclear matrix protein extraction protocols have been effective in identifying diagnostic NM protein markers for bladder cancer detection (7). Thus, the isolation of cisplatin DNA-cross-linked proteins is a complementary approach to these nuclear matrix extraction protocols, which may also be useful in the detection of additional nuclear matrix proteins for cancer diagnosis.
KeywordsHuman Breast Cancer Cell Line Nuclear Matrix Guanidine Hydrochloride Dialysis Tubing Nuclear Matrix Protein
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