Detection of DNA cross-links in tumor cells with the ethidium bromide fluorescence assay

✍ Scribed by S. de Jong; J. G. Zijlstra; H. Timmer-Bosscha; N. H. Mulder; E. G. E. de Vries

• Publisher: John Wiley and Sons
• Year: 1986
• Tongue: French
• Weight: 459 KB
• Volume: 37
• Category: Article
• ISSN: 0020-7136
• DOI: 10.1002/ijc.2910370414

✦ Synopsis

Until now the fluorescence assay with ethidium bromide has only been used on pure DNA. This assay depends on the difference in fluorescence between single-and double-stranded D N A (dsDNA). Cross-links in D N A are measured by the return of fluorescence of dsDNA after heat denaturation at PH 12. Under these conditions denatured D N A gives very low fluorescence. In the present study this assay was applied to tumor cells. The mouse Ehrlich ascites tumor cell line (EAT). and a human small-cell carcinoma line (GLC.+) were incubated for 4 hr at 37OC, with the cross-linking agent cis-diamino-dichloro platinum (cDDP). The Samples of whole cells were thereafter resuspended in potassium phosphate buffer with 10 m n EDTA, 4n NaCI, 0.1% Sarkosyl PH 7.2, for 16 hr at 37OC. Measurements were performed with a spectrofluorometer with excitation wavelength 525 nm, emission wavelength 580 nm. There was a linear relationship for cDDP concentrations of 0-150 PM and the extent of D N A cross-links in EAT (r = 0.958). In GLC4 there was a linear relationship at low cDDP concentrations of 0-50 ~L M (r = 0.968) while between 50 and 150 PM a plateau was reached. RNase added to the lysate of whole cells had no influence on the extent of crosslinks. This assay was compared with the alkaline elution assay, and results were identical.

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