EFFECTS OF CYCLOPHOSPHAMIDE ON THE MITHRAMYCIN-DNA FLUORESCENCE OF HUMAN LYMPHOMA-CELLS - POSSIBLE RESULT OF GUANINE ALKYLATION

Chicken erythrocytes can be used as an internal standard in the flow microfluorometric determination of cellular DNA content distribution. This standard controls for both instrumental and staining variation. The ratio between the G1 model channel of a tumor cell sample to that of the internal standard has been shown to correlate well with DNA mass as determined biochemically. After treatment with the alkylating agent, cyclophosphamide, changes in the mithramycin fluorescence ratio were seen in L 1210 leukemia in vivo. This observation has now also been made in two patients with lymphoma who received a single dose of cyclophosphamide. In one patient with undifferentiated lymphoma of the bone marrow, the fluorescence increased rapidly to a maximum at 24 hr, and returned to that of his peripheral lymphocytes 13 days later, with the reappearance of morphologically normal bone marrow cells. In the second patient, with lymphoblastic lymphoma cells in a pleural effusion, the fluorescence increased slowly to a maximum at 72 hr and returned to pretreatment tumor cell levels by day 6. Mithramycin binding is known to depend not only on a stoichiometric concentration of Mg++, but also upon deoxyguanosine residues in DNA, especially the 2-amino group of guanine. Alkylating agents, such as cyclophosphamide, most commonly alkylate guanine in DNA, as well as creating DNA cross-links. This could explain the effects of alkylation on mithramycin-DNA fluorescence intensity, and raises the possibility that fluorescent probes could be used to detect specific changes in macromolecular structure. Clinically, measurement of fluorescence intensity could be developed into a rapid way to quantify cell damage due to alkylation which would be particularly useful in assessing the response of slowly growing tumors.