Advertisement

Isolation of Proteins Cross-linked to DNA by Cisplatin

  • Virginia A. Spencer
  • James R. Davie
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

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.

Keywords

Human Breast Cancer Cell Line Nuclear Matrix Guanidine Hydrochloride Dialysis Tubing Nuclear Matrix Protein 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Foka, M. and Paoletti, J. (1986) Interaction of cis-diamminedichloroplatinum (II) to chro-matin. Biochem. Pharmacol. 35, 3283–3291.Google Scholar
  2. 2.
    Davie, J. R., Samuel, S. K., Spencer, V. A., Bajno, L., Sun, J. M., Chen, H. Y., and Holth, L. T. (1998) Nuclear matrix: application to diagnosis of cancer and role in transcription and modulation of chromatin structure. Gene Ther. Mol. Biol. 1, 509–528.Google Scholar
  3. 3.
    Ferraro, A., Eufemi, M., Cervoni, L., Altieri, F., and Turano, C. (1995) DNA-nuclear matrix interactions analyzed by crosslinking reactions in intact nuclei from avian liver, Acta Biochim. Pol. 42, 145–151.PubMedGoogle Scholar
  4. 4.
    Ferraro, A., Cervoni, L., Eufemi, M., Altieri, F., and Turano, C. (1996) A comparison of DNA-protein interactions in intact nuclei from avian liver and erythrocytes: a crosslinking study. J. Cell. Biochem. 62, 495–505.PubMedCrossRefGoogle Scholar
  5. 5.
    Samuel, S. K., Spencer, V. A., Bajno, L., Sun, J. M., Holth, L. T., Oesterreich, S., and Davie, J. R. (1998) In situ crosslinking by cisplatin of nuclear matrix-bound transcription factors to nuclear DNA of human breast cancer cells. Cancer Res. 58, 3004–3008.PubMedGoogle Scholar
  6. 6.
    Spencer, V. A., Samuel, S. K., and Davie, J. R. (2000) Nuclear matrix proteins associated with DNA in situ in hormone-dependent and hormone-independent human breast cancer cell lines. Cancer Res. 60, 288–292.PubMedGoogle Scholar
  7. 7.
    Konety, B. R., Nguyen, T. S., Brenes, G., Sholder, A., Lewis, N., Bastacky, S., et al. (2000) Clinical usefulness of the novel marker BLCA-4 for the detection of bladder cancer. J. Urol. 164, 634–639.PubMedCrossRefGoogle Scholar
  8. 8.
    Ferraro, A., Grandi, P., Eufemi, M., Altieri, F., Cervoni, L., and Turano, C. (1991) The presence of N-glycosylated proteins in cell nuclei. Biochem. Biophys. Res. Commun. 178, 1365–1370.PubMedCrossRefGoogle Scholar
  9. 9.
    Lippard, S. J. (1982) New chemistry of an old molecule: cis-[Pt(NH3)2Cl2]. Science 218, 1075–1082.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2002

Authors and Affiliations

  • Virginia A. Spencer
    • 1
  • James R. Davie
    • 2
  1. 1.Manitoba Institute of Cell BiologyManitobaCanada
  2. 2.Manitoba Institute of Cell BiologyWinnipegCanada

Personalised recommendations