HIGH-MOLECULAR-WEIGHT DNA FRAGMENTATION - A CRITICAL EVENT IN NUCLEOSIDE ANALOG-INDUCED APOPTOSIS IN LEUKEMIA-CELLS

Cleavage of DNA into internucleosomal fragments is one of the characte ristics of apoptosis, However, searches for in vivo evidence of nucleo somal DNA fragmentation in leukemia cells freshly obtained from patien ts during chemotherapy frequently failed to reveal nucleosomal multime rs (DNA ladders), It is not clear whether this type of DNA cleavage is an essential event in drug-induced apoptosis and thus a denominator o f cell killing, or whether the internucleosomal DNA fragments are mere ly the by-products of the apoptotic process, Here, me report our inves tigation into the role of DNA fragmentation in apoptotic cell death in duced by anticancer nucleoside analogues, both in cell culture and in leukemia patients undergoing chemotherapy, Using a 5'-end DNA-labeling technique and pulsed field gel electrophoresis, we detected fragmenta tion of DNA in two distinct size classes, internucleosomal and high mo lecular weight (predominantly 50 kb) DNA fragments, in a human leukemi a cell line exposed to the nucleoside analogues fludarabine and gemcit abine, We further demonstrated that the two types of DNA fragmentation were separate events, distinguishable by their requirements for Ca2and responses to phorbol ester treatment, The drug-treated cells under went morphological changes of apoptosis even after internucleosomal DN A fragmentation was selectively inhibited by intracellular Ca2+ chelat ion, or by treatment with phorbol ester, In contrast, neither apoptoti c morphology nor internucleosomal DNA fragmentation was observed when the high molecular weight DNA fragmentation was blocked by inhibition of nucleoside analogue incorporation into DNA, These results suggest t hat cleavage of DNA into large fragments may be an initial event that is critical for drug-induced apoptosis, whereas activation of a Ca2+-d ependent endonuclease to cleave DNA at internucleosomal sites is not a n absolute requirement for the execution of the apoptotic cell death p rogram, Further studies of leukemic lymphocytes obtained from 9 patien ts with chronic lymphocytic leukemia during therapy with fludarabine r evealed high molecular weight DNA fragmentation, which was correlated with a decrease of peripheral lymphocytes in 6 patients, whereas only 1 of the 15 patients evaluated for nucleosomal DNA fragments showed th e DNA ladders. These results indicate that high molecular weight DNA f ragmentation occurs in vivo, and may be correlated with the cytotoxic action of the anticancer drugs, Further study of the association of hi gh molecular weight DNA fragmentation with clinical response to chemot herapy is warranted.