The effect of prolonged incubations and heat denaturation on melphalan-induced DNA cross-links as measured by the ethidium bromide fluorescence assay.
Th ethidium bromide fluorescence assay detects DNA interstrand crosslinks following heat denaturation of DNA on the basis of a 20-25 fold enhancement of ethidium bromide fluorescence in the presence of double stranded DNA. This assay has been utilized to detect DNA cross-links produced by melphalan in lymphocytes from chronic lymphocytic leukemia patients. The percentage of DNA cross-links (C) in these cells did not vary linearly with the concentration of melphalan, possibly as a result of DNA fragmentation during a 16 h lysis to degrade RNA. In order to investigate this, DNA was exposed to melphalan and then the Ct was determined immediately after maximal DNA cross-link formation or after a 16 h incubation. The additional incubation period did not alter the linear relationship between Ct and melphalan concentration. Further, the DNA cross-links produced by melphalan does not appear to be heat labile since varying the heat denaturation period from 5 to 15 min had no effect on Ct. These results suggest that this assay can accurately quantitate the percentage of DNA cross-links produced by melphalan and that the curvilinear relationship between Ct and melphalan concentration in malignant human lymphocytes is not due to the ethidium bromide fluorescence assay.
Panasci
L
L
Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada.
McQuillan
A
A
Cohen
J
J
Zackon
I
I
D000818:Animals; D004247:DNA / Q000187:drug effects* / Q000378:metabolism; D004249:DNA Damage; D004305:Dose-Response Relationship, Drug; D004996:Ethidium; D005399:Fishes; D005453:Fluorescence; D006358:Hot Temperature; D006801:Humans; D008297:Male; D008558:Melphalan / Q000494:pharmacology*; D009691:Nucleic Acid Denaturation; D013997:Time Factors
D004247:DNA; D004996:Ethidium; D008558:Melphalan